Fusion proteins and polynucleotides encoding gelonin sequences

ABSTRACT

The present invention provides purified and isolated polynucleotides encoding Type I ribosome-inactivating proteins (RIPS) and analogs of the RIPs having a cysteine available for disulfide bonding to targeting molecules. Vectors comprising the polynucleotides and host cells transformed with the vectors are also provided. The RIPs and RIP analogs are particularly suited for use as components of cytotoxic therapeutic agents of the invention which include gene fusion products and immunoconjugates. Cytotoxic therapeutic agents or immunotoxins according to the present invention may be used to selectively eliminate any cell type to which the RIP component is targeted by the specific binding capacity of the second component of the agent, and are suited for treatment of diseases where the elimination of a particular cell type is a goal, such as autoimmune disease, cancer and graft-versus-host disease.

This is a continuation of U.S. application Ser. No. 09/136,389, filedAug. 18, 1998, now U.S. Pat. No. 6,146,850, which is a continuation ofU.S. application Ser. No. 08/646,360, filed May 13, 1996, now U.S. Pat.No. 5,837,491, which is the U.S. National Phase of PCT/US94/05348,internationally filed May 12, 1994, which is a continuation-in-part ofU.S. application Ser. No. 08/064,691, filed May 12, 1993 (nowabandoned), which is a continuation-in-part of U.S. application Ser. No.07/988,430, filed Dec. 9, 1992 (now U.S. Pat. No. 5,416,202), which is acontinuation-in-part of U.S. application Ser. No. 07/901,707 filed Jun.19, 1992 (now U.S. Pat. No. 5,376,546), which is a continuation-in-partof U.S. application Ser. No. 07/787,567, filed Nov. 4, 1991 (nowabandoned).

FIELD OF THE INVENTION

The present invention generally relates to materials useful ascomponents of cytotoxic therapeutic agents. More particularly, theinvention relates to ribosome-inactivating proteins, to analogs ofribosome-inactivating proteins, to polynucleotides encoding suchproteins and analogs, some of which are specifically modified forconjugation to targeting molecules, and to gene fusions ofpolynucleotides encoding ribosome-inactivating proteins topolynucleotides encoding targeting molecules.

BACKGROUND

Ribosome-inactivating proteins (RIPs) comprise a class of proteins whichis ubiquitous in higher plants. However, such proteins have also beenisolated from bacteria. RIPs are potent inhibitors of eukaryotic proteinsynthesis. The N-glycosidic bond of a specific adenine base ishydrolytically cleaved by RIPs in a highly conserved loop region of the28S rRNA of eukaryotic ribosomes thereby inactivating translation.

Plant RIPs have been divided into two types. Stirpe et al., FEBS Lett.,195(1,2):1-8 (1986). Type I proteins each consist of a single peptidechain having ribosome-inactivating activity, while Type II proteins eachconsist of an A-chain, essentially equivalent to a Type I protein,disulfide-linked to a B-chain having cell-binding properties. Gelonin,dodecandrin, tricosanthin, tricokirin, bryodin, Mirabilis antiviralprotein (MAP), barley ribosome-inactivating protein (BRIP), pokeweedantiviral proteins (PAPS), saporins, luffins, and momordins are examplesof Type I RIPs; whereas Ricin and abrin are examples of Type II RIPs.

Amino acid sequence information is reported for variousribosome-inactivating proteins. It appears that at least the tertiarystructure of RIP active sites is conserved among Type I RIPs, bacterialRIPs, and A-chains of Type II RIPs. In many cases, primary structurehomology is also found. Ready et al., J. Biol. Chem.,259(24):15252-15256 (1984) and other reports suggest that the two typesof RIPs are evolutionarily related.

Type I plant ribosome-inactivating proteins may be particularly suitedfor use as components of cytotoxic therapeutic agents. A RIP may beconjugated to a targeting agent which will deliver the RIP to aparticular cell type in vivo in order to selectively kill those cells.Typically, the targeting agent (e.g., an antibody) is linked to thetoxin by a disulfide bond which is reduced in vivo allowing the proteintoxin to separate from the delivery antibody and become activeintracellularly. Another strategy for producing targeted cytotoxicproteins is to express a gene encoding a cytotoxic protein fused to agene encoding a targeting moiety. The resulting protein product iscomposed of one or more polypeptides containing the cytotoxic proteinlinked to, for example, at least one chain of an antibody.

A variety of such gene fusions are discussed in Pastan et al., Science,254;1173-1177 (1991). However, these fusion proteins have beenconstructed with sequences from diphtheria toxin or Pseudomonasaeruginosa exotoxin A, both of which are ADP-ribosyltransferases ofbacterial origin. These protein toxins are reported to intoxicate cellsand inhibit protein synthesis by mechanisms which differ from those ofthe RIPs. Moreover, diphtheria toxin and exotoxin A are structurallydifferent from, and show little amino acid sequence similarity with,RIPs. In general, fusion proteins made with diphtheria toxin or exotoxinA have been immunogenic and toxic in animals, and are producedintracellularly in relatively low yield. Another strategy for producinga cytotoxic agent is to express a gene encoding a RIP fused to a geneencoding a targeting moiety. The resulting protein product is a singlepolypeptide containing a RIP linked to, for example, at least one chainof an antibody.

Because some RIPs, such as the Type I RIP gelonin, are primarilyavailable from scarce plant materials, it is desirable to clone thegenes encoding the RIPs to enable recombinant production of theproteins. It is also desirable to develop analogs of the naturalproteins which may be easily conjugated to targeting molecules whileretaining their natural biological activity because most Type I RIPshave no natural sites (i.e. available cysteine residues) for conjugationto targeting agents. Alternatively, it is desirable to develop genefusion products including Type I RIPs as a toxic moiety and antibodysubstances as a targeting moiety.

The present invention also provides novel humanized or human-engineeredantibodies and methods for producing such antibodies which may beconjugated or fused to various toxins. Such conjugations or fusions areuseful in the treatment of various disease states, including autoimmunediseases and cancer.

There are several reports relating to replacement of amino acids in amouse antibody with amino acids normally occurring at the analogousposition in the human form of the antibody. see, e.g., Junghaus, et al.,Cancer Res., 50: 1495-1502 (1990) and other publications which describegenetically-engineered mouse/human chimeric antibodies. Also by geneticengineering techniques, the genetic information from murinehypervariable complementarity determining regions (hereinafter referredto as “CDRs”) may be inserted in place of the DNA encoding the CDRs of ahuman monoclonal antibody to generate a construct encoding a humanantibody with murine CDRS. See, e.g., Jones, et al., Nature, 321:522-525 (1986).

Protein structure analysis on such “CDR-grafted” antibodies may be usedto “add back” murine residues in order to restore lost antigen-bindingcapability, as described in Queen, et al, Proc. Natl. Acad. Sci. (USA),86:10029-10033 (1989); Co, et al., Proc. Nat. Acad. Sci. (USA), 88:2869-2873 (1991). However, a frequent result of CDR-grafting is that thespecific binding acitvity of the resulting humanized antibodies may bediminished or completely abolished.

As demonstrated by the foregoing, there exists a need in the art forcloned genes encoding Type I RIPs, for analogs of Type I RIPs which maybe easily conjugated to targeting molecules, and for gene fusionproducts comprising Type I RIPs, and especially wherein such genefusions also comprise an humanized antibody portion.

SUMMARY OF THE INVENTION

The present invention provides purified and isolated polynucleotidesencoding Type I RIPs, Type I RIPs having a cysteine available fordisulfide bonding to targeting molecules and fusion products comprisingType I RIPs. Vectors comprising the polynucleotides and host callstransformed with the vectors are also provided.

A purified and isolated polynucleotide encoding natural sequence gelonin(SEQ ID NO: 11), and a host cell including a vector encoding gelonin ofthe type deposited as ATCC Accession No. 68721 are provided. Furtherprovided are a purified and isolated polynucleotide encoding naturalsequence barley ribosome-inactivating protein and a purified andisolated polynucleotide encoding momordin II.

Some of the polynucleotides mentioned above encode fusion proteins ofthe present invention comprising gelonin (SEQ ID NO: 2) or another RIPand an antibody or a fragment comprising an antigen-binding portionthereof. Several alternate forms of fusion proteins comprising geloninare contemplated herein. For example, the fusion protein may contain asingle RIP fused to a monovalent antibody binding moiety, such as a Fabor single chain antibody. Alternatively, multivalent forms of the fusionproteins may be made and may have greater activity than the monovalentforms. In preferred embodiments of the invention, gelonin may be fusedto either the carboxy or the amino terminus of the antibody orantigen-binding portion of thereof. Also in a preferred embodiment ofthe invention, the antibody or fragment thereof comprising anantigen-binding portion may be an he3 antibody, an he3-Fab, an he3 Fd,single-chain antibody, or an he3 kappa fragment. The antibody orantigen-binding portion thereof may be fused to gelonin by means of alinker peptide, preferably a peptide segment of shiga-like toxin asshown in SEQ ID NO: 56 or a peptide segment of Rabbit muscle aldolase asshown in SEQ ID NO: 57 or a human muscle aldolase, an example of whichis reported in Izzo, et al., Eur. J. Biochem, 174: 569-578 (1988),incorporated by reference herein.

Analogs of a Type I plant RIP are defined herein as non-naturallyoccurring polypeptides that share the ribosome-inactivating activity ofthe natural protein but that differ in amino acid sequence from thenatural type I RIP protein in some degree but less than they differ fromthe amino acid sequences of other Type I plant RIP. Preferred analogsaccording to the present invention are analogs of Type I plant RIPs eachhaving a cysteine available for disulfide bonding located at a positionin its amino acid sequence from the position corresponding to position251 in SEQ ID NO: 1 to the carboxyl terminal position of the analog. SEQID NO: 1 represents the amino acid sequence of ricin A-chain. Otherpreferred analogs according to the invention are Type I RIPs each havinga cysteine available for disulfide bonding at a position in the analogthat is on the surface of the protein in its natural conformation andthat does not impair native folding or biological activity of theribosome-inactivating protein. Analogs of bacterial RIPs are alsocontemplated by the present invention.

The present invention provides an analog of a Type Iribosome-inactivating protein, which analog has a cysteine available forintermolecular disulfide bonding at an amino acid position correspondingto a position not naturally available for intermolecular disulfidebonding in the Type I ribosome-inactivating protein and which cysteineis located at a position in the amino acid sequence of the analogcorresponding to position 259 in SEQ ID NO: 1 or at a position in theamino acid sequence in the analog corresponding to a position fromposition 251 in SEQ ID NO: 1 to the carboxyl terminal position of theanalog.

An analog according to the present invention may be an analog ofgelonin. In an analog of gelonin according to the present invention, thecysteine may be at a position in the analog from position 244 to thecarboxyl terminal position of the analog, more preferably at a positionin the analog from position 247 to the carboxyl terminal position of theanalog, and most preferably at position 244, at position 247, or atposition 248 of the amino acid sequence of the analog. In these analogs,it is preferred that the gelonin cysteine residues at positions 44 and50 be replaced with non-cysteine residues such as alanine.

An analog according to the present invention may be an analog of barleyribosome-inactivating protein. Preferably, a cysteine in such an analogis at a position in the analog from position 256 to the carboxylterminal position, and more preferably the cysteine is at a position inthe amino acid sequence of the analog from position 260 to the carboxylterminal position of the analog. Most preferably, in these regions, thecysteine is at position 256, at position 270, or at position 277 of theamino acid sequence of the analog.

An analog according to the present invention may be an analog ofmomordin II.

Analogs according to the present invention may have a cysteine in theamino acid sequence of the analog at a position which corresponds to aposition within one amino acid of position 259 of SEQ ID NO: 1. Such ananalog may be an analog of gelonin, of barley ribosome-inactivatingprotein, or of momordin II.

The present invention also provides a polynucleotide encoding an analogof a Type I ribosome-inactivating protein, which analog has a cysteineavailable for intermolecular disulfide bonding at an amino acid positioncorresponding to a position not naturally available for intermoleculardisulfide bonding in the Type I ribosome-inactivating protein, and whichcysteine is located at a position in the amino acid sequence of theanalog from the position corresponding to position 251 in SEQ ID NO: 1to the carboxyl terminal position of the analog. The polynucleotide mayencode an analog of gelonin, preferably an analog wherein the cysteineis at a position in the amino acid sequence of the analog from position244 to the carboxyl terminal position of the analog, more preferablywherein the cysteine is at a position in the analog from position 247 tothe carboxyl terminal position of the analog, and most preferably thecysteine is at position 244, at position 247 or at position 248 of theamino acid sequence of the analog. It is preferred that a polynucleotideaccording to the present invention encode a gelonin analog wherein thenative gelonin cysteine residues at positions 44 and 50 are replacedwith non-cysteine residues, such as alanine.

A polynucleotide according to the present invention may encode an analogof barley ribosome-inactivating protein, preferably an analog whereinthe cysteine is at a position in the analog from position 256 to thecarboxyl terminal position of the analog, more preferably wherein thecysteine is at a position in the analog from position 260 to thecarboxyl terminal position of the analog, and most preferably whereinthe cysteine is at position 256, at position 270 or at position 277 ofthe amino acid sequence of the analog.

A polynucleotide according to the present invention may encode an analogof mormordin II.

The present invention provides a vector including a polynucleotideencoding an analog of a Type I ribosome-inactivating protein, whichanalog has a cysteine available for intermolecular disulfide bonding ata amino acid position corresponding to a position not naturallyavailable for intermolecular disulfide bonding in the Type Iribosome-inactivating protein and which cysteine is located at aposition in the amino acid sequence of the analog from the positioncorresponding to position 251 in SEQ ID NO: 1 to the carboxyl terminalposition of the analog.

The present invention further provides a host cell including a DNAvector encoding an analog of a Type I ribosome-inactivating protein,which analog has a cysteine available for intermolecular disulfidebonding at an amino acid position corresponding to a position notnaturally available for intermolecular disulfide bonding in the Type Iribosome-inactivating protein and which cysteine is located at aposition in the amino acid sequence of the analog from the positioncorresponding to position 251 in SEQ ID NO: 1 to the carboxyl terminalposition of the analog. In such a host cell the vector may encode ananalog of gelonin, especially an analog wherein the cysteine is atposition 247 of the amino acid sequence of the analog, such as in thehost cell deposited as ATCC Accession No. 69009.

A host cell according to the present invention may include a vectorencoding barley ribosome-inactivating protein, especially preferred is ahost cell containing a BRIP analog wherein the cysteine is at position277, such as in the host cell deposited on Oct. 2, 1991 with theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852 as ATCC Accession No. 68722. Particularly preferred areprokaryotic host cells because such cells may be less sensitive to theaction or RIPs as compared to eukaryotic cells.

The present invention also provides an agent toxic to a cell includingan analog of a Type I ribosome-inactivating protein linked by adisulfide bond through a cysteine to a molecule which specifically bindsto the cell, which cysteine is at an amino acid position in the analogcorresponding to a position not naturally available for intermoleculardisulfide bonding in the Type I ribosome-inactivating protein and whichcysteine is located in the amino acid sequence of the analog from theposition corresponding to position 251 in SEQ ID NO: 1 to the carboxylterminal position of the analog. The agent may include an analog ofgelonin, preferably an analog wherein the cysteine is at a position inthe analog from position 247 to the carboxyl terminal position of theanalog, and more preferably wherein the cysteine is at position 247 or248 of the amino acid sequence of analog. An agent including an analogwherein the native gelonin cysteine residues at positions 44 and 50 arereplaced with non-cysteine residues, such as alanine is preferred.

An agent according to the present invention may include an analog ofbarley ribosome-inactivating protein, preferably an analog wherein thecysteine is at a position in the analog from position 260 to thecarboxyl terminal position of the analog, more preferably wherein thecysteine is at a position in the analog from position 270 to thecarboxyl terminal position of the analog, and most preferably whereinthe cysteine is at position 256, at position 270 or at position 277 ofthe amino acid sequence of the analog.

An agent according to the present invention may include an analog ofmomordin II.

The present invention provides an agent wherein the Type Iribosome-inactivating protein is linked to an antibody, particularly toan H65 antibody or to an antibody fragment, more particularly to anantibody fragment selected from the group consisting of chimeric andhuman engineered antibody fragments, and most particularly to a Fabantibody fragment, a Fab′ antibody fragment or a F(ab′)₂ antibodyfragment. It is highly preferred that an agent according to the presentinvention include a chimeric or human engineered antibody fragmentselected from the group consisting of a Fab antibody fragment, a Fab′antibody fragment and a F(ab′)₂ antibody fragment.

A method according to the present invention for preparing an analog of aType I ribosome-inactivating protein includes the step of expressing ina suitable host cell a polynucleotide encoding a Type Iribosome-inactivating fusion protein or type I RIP (especially gelonin)having a cysteine available for intermolecular disulfide bondingsubstituted (e.g., by site-directed mutagenesis of the natural DNAsequence encoding the RIP or by chemical synthesis of a DNA sequenceencoding the RIP analog) at an amino acid position corresponding to aposition not naturally available for intermolecular disulfide bonding inthe Type I ribosome-inactivating protein, which cysteine is located at aposition in the amino acid sequence of the analog from the positioncorresponding to position 251 in SEQ ID NO: 1 to the carboxyl terminalposition of the analog.

A product according to the present invention may be a product of amethod including the step of expressing in a suitable host cell apolynucleotide encoding a Type I ribosome-inactivating protein having acysteine available for intermolecular disulfide bonding substituted atan amino acid position corresponding to a position not naturallyavailable for intermolecular disulfide bonding in the Type Iribosome-inactivating protein, which cysteine is located at a positionin the amino acid sequence of the analog from the position correspondingto position 251 in SEQ ID NO: 1 to the carboxyl terminal position of theanalog.

The present invention provides a method for preparing an agent toxic toa cell including the step of linking an analog of a Type Iribosome-inactivating protein through a cysteine to a molecule whichspecifically binds to the cell, which analog has the cysteine at anamino acid position corresponding to a position not naturally availablefor intermolecular disulfide bonding in the Type I ribosome-inactivatingprotein and which cysteine is located at a position in the amino acidsequence of the analog from the position corresponding to position 251in SEQ ID NO: 1 to the carboxyl terminal position of the analog.

According to the present invention, a method for treating a disease inwhich elimination of particular cells is a goal may include the step ofadministering to a patient having the disease a therapeuticallyeffective amount of an agent toxic to the cells including a type I RIP(especially gelonin fused to or an analog of a Type Iribosome-inactivating protein linked through a cysteine to a moleculewhich specifically binds to the cell, the analog having the cysteine atan amino acid position corresponding to a position not naturallyavailable for intermolecular disulfide bonding in the Type Iribosome-inactivating protein and the cysteine being located at aposition in the amino acid sequence of the analog from the positioncorresponding to position 251 in SEQ ID NO: 1 to the carboxyl terminalposition of the analog.

Useful target cells for immunotoxins of the present invention include,but are not limited to, cells which are pathogenic, such as cancercells, autoimmune cells, and virally-infected cells. Such pathogeniccells may be targeted by antibodies or other targeting agents of theinvention which are joined, either by genetic engineering techniques orby chemical cross-linking, to an RIP. Specifically useful targetsinclude tumor-associated antigens (e.g., on cancer cells), celldifferentiation markers (e.g., on autoimmune cells), parasite-specificantigens, bacteria-specific antigens, and virus-specific antigens.

The present invention also provides an analog of a Type Iribosome-inactivating protein, wherein the analog has a cysteineavailable for intermolecular disulfide bonding located at an amino acidposition corresponding to a position not naturally available forintermolecular disulfide bonding in the Type I ribosome-inactivatingprotein and corresponding to a position on the surface of ricin A-chainin its natural conformation, and wherein the analog retains theribosome-inactivating activity of the Type I ribosome-inactivatingprotein.

Such a fusion protein or an analog may be a fusion protein or an analogwherein the Type I ribosome inactivating protein is gelonin, and theanalog is preferably an analog of gelonin wherein the cysteine is atposition 10 of the amino acid sequence of the analog as encoded in avector in a host cell deposited with the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852 as ATCC AccessionNo. 69008 on Jun. 9, 1992. Other such gelonin analogs include thosewherein the cysteine is at a position 60, 103, 146, 184 or 215 in theamino acid sequence of the gelonin analog. It is preferred that thegelonin cysteine residues at positions 44 and 50 be replaced withnon-cysteine residues such as alanine in these analogs.

The present invention further provides an analog of a Type Iribosome-inactivating protein wherein the analog includes only a singlecysteine. Such an analog may be an analog of gelonin and is preferablyan analog wherein the single cysteine is at position 10, position 44,position 50 or position 247 in the amino acid sequence of the analog,but the cysteine may be located at other positions defined by theinvention as well.

The present invention provides a polynucleotide encoding an analog of aType I ribosome-inactivating protein, wherein the analog has a cysteineavailable for intermolecular disulfide bonding located at an amino acidposition corresponding to a position not naturally available forintermolecular disulfide bonding in the Type I ribosome-inactivatingprotein and corresponding to a position on the surface of ricin A-chainin its natural conformation, and wherein the analog retainsribosome-inactivating activity of the Type I ribosome-inactivatingprotein.

According to the present invention, a method for preparing an analog ofa Type I ribosome-inactivating protein may include the step ofexpressing in suitable host cell a polynucleotide encoding a Type Iribosome-inactivating protein having a cysteine available forintermolecular disulfide bonding substituted at an amino acid positioncorresponding to a position not naturally available for disulfidebonding in the Type I ribosome-inactivating protein, the cysteine islocated at a position corresponding to an amino acid position on thesurface of ricin A-chain in its natural conformation and which analogretains ribosome-inactivating activity of the Type Iribosome-inactivating protein.

The present invention provides an agent toxic to a cell including ananalog of a Type I ribosome-inactivating protein linked by a disulfidebond through a cysteine to a molecule which specifically binds to thecell, wherein the analog has a cysteine available for intermoleculardisulfide bonding located at an amino acid position corresponding to aposition not naturally available for intermolecular disulfide bonding inthe Type I ribosome-inactivating protein and corresponding to a positionon the surface of ricin A-chain in its natural conformation, and whereinthe analog retains ribosome-inactivating activity of the Type Iribosome-inactivating protein.

A method according to the present invention for preparing an agent toxicto a cell may include the step of linking an analog of a Type Iribosome-inactivating protein through a cysteine to a molecule whichspecifically binds to the cell, which analog has a cysteine availablefor intermolecular disulfide bonding located at an amino acid positioncorresponding to a position not naturally available for intermoleculardisulfide bonding in the Type I ribosome-inactivating protein andcorresponding to a position on the surface of ricin A-chain in itsnatural conformation, and which analog retains ribosome-inactivatingactivity of the Type I ribosome-inactivating protein.

A method according to the present invention for treating a disease inwhich elimination of particular cells is a goal includes the step ofadministering to a patient having the disease a therapeuticallyeffective amount of an agent toxic to the cells wherein the agentincludes a type I RIP fused to or an analog of a Type Iribosome-inactivating protein linked by a disulfide bond through acysteine to a molecule which specifically binds to the cell, whichanalog has a cysteine available for intermolecular disulfide bondinglocated at an amino acid position corresponding to a position notnaturally available for intermolecular disulfide bonding in the Type Iribosome-inactivating protein and corresponding to a position on thesurface of ricin A-chain in its natural conformation, and which analogretains ribosome-inactivating activity of the Type Iribosome-inactivating protein.

The RIP analogs of the invention are particularly suited for use ascomponents of cytotoxic therapeutic agents. Cytotoxic agents accordingto the present invention may be used in viva to selectively eliminateany cell type to which the RIP component is targeted by the specificbinding capacity of the second component. To form cytotoxic agents, RIPanalogs may be conjugated to monoclonal antibodies, including chimericand CDR-grafted antibodies, and antibody domains/fragments (e.g., Fab,Fab′, F(ab′)₂, single chain antibodies, and Fv or single variabledomains). Analogs of RIPs conjugated to monoclonal antibodiesgenetically engineered to include free cysteine residues are also withinthe scope of the present invention. Examples of Fab′ and F(ab′)₂fragments useful in the present invention are described in co-pending,co-owned U.S. patent application Ser. No. 07/714,175, filed Jun. 14,1991 and in International Publication No. WO 89/00999 published on Feb.9, 1989, which are incorporated by reference herein.

The RIP analogs of the invention may preferably be conjugated or fusedto humanized or human engineered antibodies, such as the he3 antibodydescribed herein. Such humanized antibodies may be constructed frommouse antibody variable domains, such as the mouse antibody H65 (SEQ IDNOS: 123 and 124). Specifically RIP analogs according to the presentinvention may be conjugated or fused to he3 human-engineered antibodylight and heavy chain variable regions (SEQ ID NO: 125 and 126,respectively) or fragments thereof. A cell line producing an intact he3immunoglobulin was deposited as ATCC Accession No. HB11206 with theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852.

RIPs according to the present invention may also be conjugated totargeting agents other than antibodies, for example, lectins which bindto cells having particular surface carbohydrates, hormones, lymphokines,growth factors or other polypeptides which bind specifically to cellshaving particular receptors. Immunoconjugates including RIPs may bedescribed as immunotoxins. An immunotoxin may also consist of a fusionprotein rather than an immunoconjugate.

The present invention provides gene fusions of an antigen-bindingportion of an antibody (e.g., an antibody light chain or truncated heavychain, or a single chain antibody) or any targeting agent listed in theforegoing paragraph, linked to a Type I RIP. Preferably, theantigen-binding portion of an antibody or fragment thereof comprises asingle chain antibody, a Fab fragment, or a F(ab′)₂ fragment. Activeantibodies generally have a conserved three-dimensional folding patternand it is expected that any antibody which maintains that foldingpattern will retain binding specificity. Such antibodies are expected toretain target enzymatic activity when incorporated into a fusion proteinaccording to the present invention.

It is sometimes necessary that immunotoxins comprising cytotoxiccomponents, such as RIPs, be attached to targeting agents via cleavablelinkers (i.e., disulfides, acid-sensitive linkers, and the like) inorder to allow intracellular release of the cytotoxic component. Suchintracellular release allows the cytotoxic component to functionunhindered by possible negative kinetic or steric effects of theattached antibody. Accordingly, gene fusions of the present inventionmay comprise a RIP gene fused, via a DNA segment encoding a linkerprotein as described above, to either the 5′ or the 3′ end of a geneencoding an antibody. If a linker is used, it preferably encodes apolypeptide which contains two cysteine residues participating in adisulfide bond and forming a loop which includes a protease-sensitiveamino acid sequence (e.g., a segment of E. coli shiga-like toxin as inSEQ ID NO: 56) or a segment which contains several cathepsin cleavagesites (e.g., a segment of rabbit muscle aldolase as in SEQ ID NO: 57; asegment of human muscle aldolase; or a synthetic peptide including acathepsin cleavage sites such as in SEQ ID NOs: 141 or 142). A linkercomprising cathepsin cleavage sites as exemplified herein comprises theC-terminal 20 amino acids of RMA. However, that sequence differs by onlyone amino acid from human muscle aldolase and it is contemplated thatmuscle aldolase from human or other sources may be used as a linker inthe manner described below. The Type I RIP portion of the fused genespreferably encodes gelonin, BRIP or momordin II. Also preferably, theantibody portion of the fused genes comprises sequences encoding one ofthe chains of an antibody Fab fragment (i.e., kappa or Fd) and the fusedgene is co-expressed in a host cell with the other Fab gene, or theantibody portion comprises sequences encoding a single chain antibody.

Alternatively, since fusion proteins of the present invention may be oflow (approximately 55 kDa) molecular weight while maintaining fullenzymatic activity, such fusions may be constructed without a linker andstill possess cytotoxic activity. Such low-molecular weight fusions arenot as susceptible to kinetic and steric hinderance as are the largerfusions, such as fusions involving IgG molecules. Therefore, cleavage ofthe RIP away from the fusion may not be necessary to facilitate activityof the RIP.

The present invention also provides a method for purifying a protein orimmunotoxin comprising a ribosome-inactivating protein and a portion ofan antibody including the steps of passing a solution containing theprotein through an anion exchange column; applying the flow-through to aprotein G column; and eluting the protein from the protein G column. Themethod may further comprise the steps of introducing the flow-through ofthe anion exchange column into a cation exchange column; exposing thecation exchange column to an eluent effective to elute said protein; andthen applying the eluted protein to a protein G column, rather thanapplying the anion exchange column flow-through directly to a protein Gcolumn.

Immunotoxins according to the present invention, includingimmunoconjugates and fusion proteins (immunofusions), are suited fortreatment of diseases where the elimination of a particular cell type isa goal, such as autoimmune disease, cancer, and graft-versus-hostdisease. The immunotoxins are also suited for use in causingimmunosuppression and in treatment of infections by viruses such as theHuman Immunodeficiency Virus.

Specifically illustrating polynucleotide sequences according to thepresent invention are the inserts in the plasmid pING3731 in E. coliMC1061 (designated strain G274) and in the plasmid pING3803 in E. coliE104 (designated strain G275), both deposited with the American TypeCulture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md., on Oct.2, 1991, and assigned ATCC Accession Nos. 68721 and 68722, respectively.Additional polynucleotide sequences illustrating the invention are theinserts in the plasmid pING3746 in E. coli E104 (designated strain G277)and in the plasmid pING3737 in E. coli E104 (designated strain G276),which were both deposited with the ATCC on Jun. 9, 1992, and wererespectively assigned Accession Nos. 69008 and 69009. Still otherpolynucleotide sequences illustrating the invention are the inserts inthe plasmid pING3747 in E. coli E104 (designated strain G278), in theplasmid pING3754 in E. coli E104 (designated strain G279), in theplasmid pING3758 in E. coli E104 (designated strain G280) and in theplasmid pING3759 in E. coli E104 (designated strain G281), whichplasmids were all deposited with the ATCC on Oct. 27, 1992 and wereassigned ATCC Accession Nos. 69101, 69102, 69103 and 69104,respectively.

As noted above, RIPs may preferably be conjugated or fused to humanizedor human-engineered antibodies, such as he3. Thus, the present inventionalso provides novel proteins comprising a humanized antibody variabledomain which is specifically reactive with an human CD5 celldifferentiation marker. Specifically, the present invention providesproteins comprising the he3 light and heavy chain variable regions asshown in SEQ ID NOS:125 or 126, respectively. DNA encoding certain he3proteins is shown in SEQ ID NOS:72 and 71.

In a preferred embodiment of the present invention, the proteincomprising an humanized antibody variable region is an intact he3immunoglobulin deposited as ATCC HB 11206.

Also in a preferred embodiment of the invention, the protein comprisinga humanized antibody variable region is a Fab or F(ab′)₂ or Fabfragment.

Proteins according to the present invention may be made by methodstaught herein and in co-pending, co-owned U.S. patent application Ser.No. 07/808,464 by Studnicka et al. incorporated by reference herein; andmodified antibody variable domains made by such methods may be used intherapeutic administration to humans either alone or as part of animmunoconjugate as taught in co-owned, co-pending U.S. patentapplication Ser. No. 07/787,567 by Better et al.

The present invention also provides methods for preparing a modifiedantibody variable domain useful in preparing immunotoxins andimmunofusions by determining the amino acids of a subject antibodyvariable domain which may be modified without diminishing the nativeaffinity of the domain for antigen while reducing its immunogenicitywith respect to a heterologous species. As used herein, the term“subject antibody variable domain” refers to the antibody upon whichdeterminations are made. The method includes the following steps:determining the amino acid sequence of a subject light chain and asubject heavy chain of a subject antibody variable domain to bemodified; aligning by homology the subject light and heavy chains with aplurality of human light and heavy chain amino acid sequences;identifying the amino acids in the subject light and heavy chainsequences which are least likely to diminish the native affinity of thesubject variable domain for antigen while, at the same time, reducingits immunogenicity by selecting each amino acid which is not in aninterface region of the subject antibody variable domain and which isnot in a complementarity-determining region or in an antigen-bindingregion of the subject antibody variable domain, but which amino acid isin a position exposed to a solvent containing the antibody; changingeach residue identified above which aligns with a highly or a moderatelyconserved residue in the plurality of human light and heavy chain aminoacid sequences if said identified amino acid is different from the aminoacid in the plurality.

Another group of sequences, such as those in FIGS. 1A and 1B may be usedto determine an alignment from which the skilled artisan may determineappropriate changes to make.

The present invention provides a further method wherein the plurality ofhuman light and heavy chain amino acid sequences is selected from thehuman consensus sequences in FIGS. 10A and 10B.

In general, human engineering according to the above methods may be usedto treat various diseases against which monoclonal antibodies generallymay be effective. However, humanized antibodies possess the additionaladvantage of reducing the immunogenic response in the treated patient.

Additional aspects and applications of the present invention will becomeapparent to the skilled artisan upon consideration of the detaileddescription of the invention which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (RTA) (SEQ ID NO: 1) with the amino acid sequence ofthe Type I ribosome-inactivating protein gelonin (SEQ ID NO: 2), whereinstarred positions indicate amino acids invariant among the ricin A-chainand the Type I RIPs;

FIG. 2 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein BRIP (SEQ ID NO: 3), whereinstarred positions indicate amino acids invariant among the ricin A-chainand the Type I RIPs;

FIG. 3 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein momordin II (MOMOII) (SEQ ID NO:4), wherein starred positions indicate amino acids invariant among thericin A-chain and the Type I RIPs;

FIG. 4 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein luffin (SEQ ID NO: 5), whereinstarred positions indicate amino acids invariant among the ricin A-chainand the Type I RIPs;

FIG. 5 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein αtrichosanthin (TRICHO) (SEQ ID NO:6), wherein starred positions indicate amino acids invariant among thericin A-chain and the Type I RIPs;

FIG. 6 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein momordin I (MOMOI) (SEQ ID NO: 7),wherein starred positions indicate amino acids invariant among the ricinA-chain and the Type I RIPs;

FIG. 7 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein Mirabilis anti-viral protein (MAP)(SEQ ID NO: 8), wherein starred positions indicate amino acids invariantamong the ricin A-chain and the Type I RIPs;

FIG. 8 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein pokeweed antiviral protein fromseeds (PAPS) (SEQ ID NO: 9), wherein starred positions indicate aminoacids invariant among the ricin A-chain and the Type I RIPs;

FIG. 9 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosomeinactivating protein saporin 6 (SAP6) (SEQ ID NO: 10),wherein starred positions indicate amino acids invariant among the ricinA-chain and the Type I RIPs;

FIGS. 10A and 10B are alignments of the consensus amino acid sequencesfor the subgroups of light [hK1 (SEQ ID NO: 149) (human kappa lightchain subgroup 1), hK3 (SEQ ID NO: 150) (human kappa light chainsubgroup 3), hK2 (SEQ ID NO: 151) (human kappa light chain subgroup 2),hL1 (SEQ ID NO: 152) (human lambda light chain subgroup 1), hL2 (SEQ IDNO: 153) (human lambda light chain subgroup 2), hL3 (SEQ ID NO:154)(human lambda light chain subgroup 3), hL6 (SEQ ID NO: 155) (humanlambda light chain subgroup 6), hK4 (SEQ ID NO: 156) (human kappa lightchain subgroup 4), hL4 (SEQ ID NO: 157) (human lambda light chainsubgroup 4) and hL5 (SEQ ID NO: 158) (human lambda light chain subgroup5)] and heavy chains [hH3 (SEQ ID NO: 159) (human heavy chain subgroup3), hH1 (SEQ ID NO: 160) (human heavy chain subgroup 1) and hH2 (SEQ IDNO: 161) (human heavy chain subgroup 2)], respectively, of humanantibody variable domains;

FIGS. 11A and 11B set out the nucleotide sequences of theoligonucleotides utilized in the construction of the genes encodingmodified V/J-regions of the light and heavy chains of the H65 mousemonoclonal antibody variable domain sequence $H65K-1:SEQ ID No. 117;HUH-K1:SEQ ID No. 141; HUH-K2:SEQ ID No. 142; HUH-K3:SEQ ID No. 143;HUH-K4:SEQ ID No. 121; HUH-K5:SEQ ID No. 122; HUH-G1:SEQ ID No. 144;HUH-G2:SEQ ID No. 145; HUH-G3:SEQ ID No. 137; HUH-G4:SEQ ID No. 138;HUH-G5:SEQ ID No. 139; HUH-G6:SEQ ID No. 140; H65G-2S:SEQ ID No. 146;H65-G2:SEQ ID No. 85; H65K-2S:SEQ ID No. 116; JK1-HindIII:SEQ ID No. 87;and

FIGS. 12A and 12B are alignments of human light chain consensus hK1 (SEQID No. 149) and heavy chain consensus hH1 (SEQ ID No. 160) with thelight and heavy chain sequences, respectively, of the variable domain ofhuman antibody EU (SEQ ID Nos. 162 and 166), human antibody TAC (SEQ IDNos. 163 and 167), human antibody TAC modified according to the presentinvention (prop) (SEQ ID Nos. 164 and 168) and human antibody TACmodified according to a different method (Que) (SEQ ID Nos. 165 and169).

DETAILED DESCRIPTION

Nucleotide sequences of genes encoding three plant Type I RIPs andexpression vectors containing the genes are provided by the presentinvention. A first plant RIP, gelonin, is produced by seeds of Geloniummultiflorum, a plant of the Euphorbiaceae family native to the tropicalforests of eastern Asia, while a second plant RIP, BRIP, is synthesizedby the common cereal grain barley. Momordin II, a third plant RIP, isproduced in Momordica balsamina seeds. Analogs of BRIP are also providedby the present invention. The analogs were genetically engineered toinclude a cysteine free to participate in a intermolecular disulfidebond and were conjugated to antibody molecules without non-specificchemical derivatization of the RIP with crosslinking agents.

Type I RIP analogs of the present invention offer distinct advantagesover the natural proteins for use as components of immunotoxins.Chemical treatment to introduce free sulfhydryl groups in the naturalproteins lacking free cysteines typically involves the non-selectivemodification of amino acid side chains. This non-selectivity oftenresults in antibodies conjugated to different sites on different RIPmolecules (i.e., a heterogeneous population of conjugates) and also in adecrease in RIP activity if antibodies are conjugated in or nearimportant regions of the RIP (e.g., the active site or regions involvedin translocation across cell membranes). In contrast, RIP analogsaccording to the present invention may be conjugated to a singleantibody through a disulfide bond to a specific residue of the analogresulting in reduced batch to batch variation of the immunoconjugatesand, in some cases, immunoconjugates with enhanced properties (e.g.,greater cytotoxicity or solubility).

Type I plant RIPs, as well as bacterial RIPs such as shiga andshiga-like toxin A-chains, are homologous to the ricin A-chain and areuseful in the present invention.

Type I RIPs may be defined and sites for substitution of a cysteine in aRIP may be identified by comparing the primary amino acid sequence ofthe RIP to the natural ricin A-chain amino acid sequence, the tertiarystructure of which has been described in Katzin et al., Proteins,10:251-259 (1991), which is incorporated by reference herein.

Amino acid sequence alignment defines Type I RIPs in that the ricinA-chain and the Type I plant RIPs have nine invariant amino acids incommon. Based on the ricin sequence the invariant amino acids aretyrosine₂₁, arginine₂₉, tyrosine₈₀, tyrosine₁₂₃, leucine₁₄₄, glutamicacid₁₇₇, alanine₁₇₈, arginine₁₈₀, and tryptophan₂₁₁. The ricin A-chainmay be used as a model for the three-dimensional structure of Type IRIPs. A protein lacking a cysteine available for conjugation whilehaving ribosome-inactivating activity and having the nine invariantamino acids when its primary sequence is compared to the primarysequence of the ricin A-chain [according to the alignment algorithm ofMyers et al., CABIOS COMMUNICATIONS, 4(1):11-17 (1988), implemented bythe PC/GENE program PALIGN (Intelligenetics, Inc., Mountain View,Calif.) and utilizing the Dayhoff Mutation Data Matrix (MDM-78) asdescribed in Schwartz et al., pp. 353-358 in Atlas of Protein Sequenceand Structure, 5 Supp. 3, National Biomedical Research Foundation,Washington, D.C. (1978)] is defined as a Type I RIP herein and isexpected to be useful in the present invention. “Corresponding” refersherein to amino acid positions which align when two amino acid sequencesare compared by the strategy of Myers et al., supra.

The primary amino acid sequences of the Type I RIPs:gelonin, BRIP,momordin II, luffin [see Islam et al., Agricultural Biological Chem.,54(5):1343-1345 (199)], αtrichosanthin [see Chow et al., J. Biol. Chem.,265:8670-8674 (1990)], momordin I [see Ho et al., BBA, 1088:311-314(1991)], Mirabilis anti-viral protein [see Habuka et al., J. Biol.Chem., 264(12):6629-6637 (1989)], pokeweed antiviral protein isolatedfrom seeds [see Kung et al., Agric. Biol. Chem., 54(12):3301-3318(1990)] and saporin [see Benatti et al., Eur. J. Biochem., 183:465-470(1989)] are individually aligned with the primary sequence of the ricinA-chain [see Halling et al., Nucleic Acids Res., 13:8019-8033 (1985)] inFIGS. 1-9, respectively, according to the algorithm of Myers et al.,supra, as specified above.

FIGS. 1-9 may be utilized to predict the amino acid positions of theType I RIPs where cysteine residues may be substituted. Preferred aminoacids for cysteine substitution are on the surface of the molecule andinclude any solvent accessible amino acids which will not interfere withproper folding of the protein if replaced with a cysteine. A region ofthe ricin A-chain comprising such amino acids is the carboxyl terminalregion. Amino acids that should be avoided for replacement are thosecritical for proper protein folding, such as proline, and those that aresolvent inaccessible. Also to be avoided are the nine amino acidsinvariant among RIPs, and the amino acids in or near regions comprisingthe active site of ricin A-chain as depicted in FIG. 6 of Katzin et al.,supra.

Therefore, a preferred region of substitution for Type I RIPs is theircarboxyl terminal region which is solvent accessible and corresponds tothe carboxyl terminal region where Type II RIP A-chains and B-chains arenaturally linked by a disulfide bond. As shown in the examples, acysteine may be substituted in positions in the amino acid sequence of aType I RIP from the position corresponding to position 251 in SEQ ID NO:1 to the carboxyl terminal position of said Type I RIP, resulting in RIPanalogs which retain enzymatic activity and gain disulfide cross-linkingcapability. One preferred cysteine substitution position is near theposition which corresponds to the cysteine at position 259 in the ricinA-chain.

For purposes of the present invention, immunotoxins comprise a class ofcompounds of which toxin-antibody fusions and immunoconjugates areexamples. Immunotoxins are particularly suited for use in treatment ofhuman autoimmune diseases and in the treatment of diseases in whichdepletion of a particular cell type is a goal, such as cancer. Forexample, treatment of autoimmune diseases with immunotoxins is describedin International Publication No. WO89/06968 published Aug. 10, 1989,which is incorporated by reference herein.

In any treatment regimen, the immunotoxins may be administered to apatient either singly or in a cocktail containing two or moreimmunotoxins, other therapeutic agents, compositions, or the like,including, but not limited to, immunosuppressive agents,tolerance-inducing agents, potentiators and side-effect relievingagents. Particularly preferred are immunosuppressive agents useful insuppressing allergic reactions of a host. Preferred immunosuppressiveagents include prednisone, prednisolone, DECADRON (Merck, Sharp & Dohme,West Point, Pa.), cyclophosphamide, cyclosporine, 6-mercaptopurine,methotrexate, azathioprine and i.v. gamma globulin or their combination.Preferred potentiators include monensin, ammonium chloride, perhexiline,verapamil, amantadine and chloroquine. All of these agents areadministered in generally-accepted efficacious dose ranges such as thosedisclosed in the Physician's Desk Reference, 41st Ed., Publisher EdwardR. Barnhart, New Jersey (1987). Patent Cooperation Treaty (PCT) U.S.patent application WO 89/069767 published on Aug. 10, 1989, disclosesadministration of an immunotoxin as an immunosuppressive agent and isincorporated by reference herein.

Immunotoxins of the present invention may be formulated into either aninjectable or topical preparation. Parenteral formulations are known andare suitable for use in the invention, preferably for intramuscular orintravenous administration. The formulations containingtherapeutically-effective amounts of immunotoxins are either sterileliquid solutions, liquid suspensions, or lyophilized versions, andoptionally contain stabilizers or excipients. Lyophilized compositionsare reconstituted with suitable diluents, e.g., water for injection,saline, 0.3% glycine and the like, at a level of about from 0.01 mg/kgof host body weight to 10 mg/kg where the biological activity is lessthan or equal to 20 ng/ml when measured in a reticulocyte lysate assay.Typically, the pharmaceutical compositions containing immunotoxins ofthe present invention are administered in a therapeutically effectivedose in a range of from about 0.01 mg/kg to about 5 mg/kg of thepatient. A preferred, therapeutically effective dose of thepharmaceutical composition containing immunotoxins of the invention isin a range of from about 0.01 mg/kg to about 0.5 mg/kg body weight ofthe patient administered over several days to two weeks by dailyintravenous infusion, each given over a one hour period, in a sequentialpatient dose-escalation regimen.

Immunotoxin compositions according to the invention may be formulatedinto topical preparations for local therapy by including atherapeutically effective concentration of immunotoxin in adermatological vehicle. The amount of immunotoxin to be administered,and the immunotoxin concentration in the topical formulations, dependupon the vehicle selected, the clinical condition of the patient, thesystemic toxicity and the stability of the immunotoxin in theformulation. Thus, a physician knows to employ the appropriatepreparation containing the appropriate concentration of immunotoxins inthe formulation, as well as the appropriate amount of formulation toadminister depending upon clinical experience with the patient inquestion or with similar patients. The concentration of immunotoxin fortopical formulations is in the range of greater than from about 0.1mg/ml to about 25 mg/ml. Typically, the concentration of immunotoxin fortopical formulations is in the range of greater than from about 1 mg/mlto about 20 mg/ml. Solid dispersions of immunotoxins according to theinvention, as well as solubilized preparations, may be used. Thus, theprecise concentration to be used in the vehicle is subject to modestexperimental manipulation in order to optimize the therapeutic response.For example, greater than about 10 mg immunotoxin/100 grams of vehiclemay be useful with 1% w/w hydrogel vehicles in the treatment of skininflammation. Suitable vehicles, in addition to gels, are oil-in-wateror water-in-oil emulsions using mineral oils, petroleum and the like.

Immunotoxins according to the invention may be optionally administeredtopically by the use of a transdermal therapeutic system [Barry,Dermatological Formulations, p. 181 (1983) and literature citedtherein]. While such topical delivery systems may be designed fortransdermal administration of low molecular weight drugs, they arecapable of percutaneous delivery. Further, such systems may be readilyadapted to administration of immunotoxin or derivatives thereof andassociated therapeutic proteins by appropriate selection of therate-controlling microporous membrane.

Topical preparations of immunotoxin either for systemic or localdelivery may be employed and may contain excipients as described abovefor parenteral administration and other excipients used in a topicalpreparation such as cosolvents, surfactants, oils, humectants,emollients, preservatives, stabilizers and antioxidants.Pharmacologically-acceptable buffers may be used, e.g., Tris orphosphate buffers. The topical formulations may also optionally includeone or more agents variously termed enhancers, surfactants, accelerants,adsorption promoters or penetration enhancers, such as an agent forenhancing percutaneous penetration of the immunotoxin or other agents.Such agents should desirably possess some or all of the followingfeatures as would be known to the ordinarily skilled artisan:pharmacological inertness, non-promotive of body fluid or electrolyteloss, compatible with immunotoxin (non-inactivating), and capable offormulation into creams, gels or other topical delivery systems asdesired.

Immunotoxins according to the present invention may also be administeredby aerosol to achieve localized delivery to the lungs. This isaccomplished by preparing an aqueous aerosol, liposomal preparation orsolid particles containing immunotoxin. Ordinarily, an aqueous aerosolis made by formulating an aqueous solution or suspension of immunotoxintogether with conventional pharmaceutically acceptable carriers andstabilizers. The carriers and stabilizers vary depending upon therequirements for the particular immunotoxin, but typically include:nonionic surfactants (Tweens, Pluronics, or polyethylene glycol);innocuous proteins like serum albumin, sorbitan esters, oleic acid,lecithin; amino acids such as glycine; and buffers, salts, sugars orsugar alcohols. The formulations may also include mucolytic agents aswell as bronchodilating agents. The formulations are sterile. Aerosolsgenerally are prepared from isotonic solutions. The particles optionallyinclude normal lung surfactants.

Alternatively, immunotoxins of the invention may be administered orallyby delivery systems such as proteinoid encapsulation as described bySteiner, et al., U.S. Pat. No. 4,925,673, incorporated by referenceherein. Typically, a therapeutically-effective oral dose of animmunotoxin according to the invention is in the range from about 0.05mg/kg body weight to about 50 mg/kg body weight per day. A preferredeffective dose is in the range from about 0.05 mg/kg body weight toabout 5 mg/kg body weight per day.

Immunotoxins according to the present invention may be administeredsystemically, rather than topically, by injection intramuscularly,subcutaneously, intrathecally or intraperitoneally or into vascularspaces, particularly into the joints, e.g., intraarticular injection ata dosage of greater than about 1 μg/cc joint fluid/day. The dose will bedependent upon the properties of the specific immunotoxin employed,e.g., its activity and biological half-life, the concentration ofimmunotoxin in the formulation, the site and rate of dosage, theclinical tolerance of the patient involved, the disease afflicting thepatient and the like, as is well within the skill of the physician.

The immunotoxins of the present invention may be administered insolution. The pH of the solution should be in the range of pH 5 to 9.5,preferably pH 6.5 to 7.5. The immunotoxin or derivatives thereof shouldbe in a solution having a suitable pharmaceutically-acceptable buffersuch as phosphate, Tris(hydroxymethyl)aminomethane-HCl or citrate andthe like. Buffer concentrations should be in the range of 1 to 100 mM.The immunotoxin solution may also contain a salt, such as sodiumchloride or potassium chloride in a concentration of 50 to 150 mM. Aneffective amount of a stabilizing agent such as an albumin, a globulin,a gelatin, a protamine or a salt of protamine may also be included, andmay be added to a solution containing immunotoxin or to the compositionfrom which the solution is prepared.

Systemic administration of immunotoxin may be made daily and isgenerally by intramuscular injection, although intravascular infusion isacceptable. Administration may also be intranasal or by othernonparenteral routes. Immunotoxins of the present invention may also beadministered via microspheres, liposomes or other microparticulatedelivery systems placed in certain tissues including blood. Topicalpreparations are applied daily directly to the skin or mucosa and arethen preferably occluded, i.e., protected by overlaying a bandage,polyolefin film or other barrier impermeable to the topical preparation.

The following Examples are illustrative of practice of the invention butare not to be construed as limiting the invention. The presentapplication is broadly organized as follows. The first portion of theapplication broadly teaches the preparation, expression and propertiesof an exemplary RIP, gelonin. A second portion of the applicationteaches the preparation of human-engineered antibodies. A third portionof the application teaches the construction and properties ofimmunoconjugates, comprising an RIP and an antibody or fragment thereofcomprising an antigen-binding portion. A forth portion of theapplication relates to the preparation and properties of immunofusionproteins comprising an RIP and an antibody or fragment thereofcomprising an antigen-binding portion. A fifth portion of theapplication teaches the preparation and properties of the RIP Barleyribosome-inactivating protein and a final aspect of the inventionprovides the preparation and properties of the RIP momordin.

Specifically, Example 1 relates to the preparation of the RIP gelonin.Construction of expression vector, comprising the gelonin gene,including expression and purification of gelonin, is taught in Example2. The assembly of gelonin genes with cysteine residues available forconjugation is taught in Example 3 and Example 4 provides results of areticulocyte lysate assay performed on gelonin.

Example 5 teaches the construction of human-engineered antibodies foruse in immunotoxins of the invention and Example 6 demonstratestransfection of he3 genes, expression of those genes, and purificationof the products thereof.

Example 7 next teaches the preparation of gelonin immunoconjugates. Theprocedures and results of whole cell kill assays are next presented inExample 8. Various properties of gelonin immunoconjugates are taught inExample 9 and Examples 10 and 11 teach the pharmacokinetics of two typesof immunoconjugates. Examples 12 and 13 teach the immunogenicity ofimmunoconjugates of the invention and the in vivo efficacy of thoseimmunoconjugates, respectively.

The construction of genes encoding gelonin immunofusions is taught inExamples 14, 15, 16, 17 and 18. Example 19 teaches alternative cathepsincleavable linkers for use in the immunofusions of the invention. Theexpression and purification of various genes encoding immunoconjugatesare presented in Example 20 and their activity properties are presentedin Example 21.

The construction of genes encoding the RIP, BRIP, and its expression andproperties are taught in Examples 22, 23, and 24.

Finally, construction of genes encoding momordin and properties ofmomordin on expression are taught in Example 25.

EXAMPLE 1

Preparation of Gelonin

The cloning of the gelonin gene according to the present inventionobviates the requirement of purifying the RIP gene product from itsrelatively scarce natural source, G. multiflorum seeds. Cloning alsoallows development of gelonin analogs which may be conjugated toantibodies without prior chemical derivatization and also allowsdevelopment of gelonin gene fusion products.

A. Preparation of RNA from G. Multiflorum Seeds

Total RNA was prepared from Gelonium seeds (Dr. Michael Rosenblum, M.D.Anderson Cancer Center, Houston, Tex.) by a modification of theprocedure for preparation of plant RNA described in Ausubel et al.,eds., Current Protocols in Molecular Biology, Wiley & Sons, 1989.Briefly, 4.0 grams of seeds were ground to a fine powder in a pre-cooled(−70° C.) mortar and pestle with liquid N₂. The powder was added to 25ml Grinding buffer (0.18M Tris, 0.09M LiCl, 4.5 mM EDTA, 1% SDS, pH 8.2)along with 8.5 ml of phenol equilibrated with TLE (0.2M Tris, 0.1M LiCl,5 mM EDTA pH8.2). The mixture was homogenized using a Polytron PT-1035(#5 setting). 8.5 ml of chloroform was added, mixed and incubated at 50°C. for 20 minutes. The mixture was centrifuged at 3000 g for 20 minutesin a rotor precooled to 4° C. and the aqueous phase was transferred to anew tube. 8.5 ml of phenol was added followed by 8.5 ml of chloroformand the mixture was recentrifuged. This extraction was repeated 3 times.The RNA in the aqueous phase was then precipitated by adding ⅓ volume 8MLiCl, and incubated at 4° C. for 16 hours. Next, the RNA was pelleted bycentrifugation for 20 minutes at 4° C. The pellet was washed with 5 mlof 2M LiCl, recentrifuged and resuspended in 2 ml of water. The RNA wasprecipitated by addition of NaOAc to 0.3M and 2 volumes of ethanol. TheRNA was stored in 70% ethanol at −70° C.

B. cDNA Preparation

cDNA was prepared from total Gelonium RNA by two methods. The firstmethod involved making a cDNA library in the bacterial expressionplasmid pcDNAII using the Librarian II cDNA Library Construction Systemkit (Invitrogen). Approximately 5 μg of total RNA was converted to firststrand cDNA with a 1:1 mixture of random primers and oligo-dT. Secondstrand synthesis with DNA polymerase I was performed as described by thesystem manufacturer. Double stranded cDNA was ligated to BstX1 linkersand size fractionated. Pieces larger than about 500 bp were ligated intothe expression vector provided in the kit. Individual vectors wereintroduced into E. coli either by transformation into high-efficiencycompetent cells or by electroporation into electrocompetent cells.Electroporation was performed with a BTX100 unit (BTX, San Diego,Calif.) in 0.56μ Flatpack cells as recommended by BTX based on themethod of Dower et al., Nucleic Acids Res., 16:6127-6145 (1988), at avoltage amplitude of 850 V and a pulse length of 5 mS. The resultinglibrary consisted of approximately 150,000 colonies.

The second method involved generating cDNA using the RNA-PCR kit sold byPerkin-Elmer-Cetus. About 100 ng of total Gelonium RNA was used astemplate for cDNA synthesis.

C. Determination of the Gelonin Protein Sequence

The partial sequence of the native gelonin protein was determined bydirect amino acid sequence analysis using automated Edman degradation asrecommended by the manufacturer using an Applied Biosystems model 470Aprotein sequencer. Proteolytic peptide fragments of gelonin (isolatedfrom the same batch of seeds as the total RNA) were sequenced.

D. Cloning of the Gelonin Gene

Three overlapping gelonin cDNA fragments were cloned and a compositegelonin gene was assembled from the three fragments.

1. Cloning Of The Fragment Encoding The Middle Amino Acids Of Gelonin InVector pING3823

Degenerate DNA primers based on the gelonin partial amino acid sequenceswere used to PCR-amplify segments of the cDNA generated withPerkin-Elmor-Cetus kit. Six primers were designed based on regions ofthe gelonin amino acid sequence where degeneracy of the primers could beminimized. Appropriate pairs of primers were tested for amplification ofa gelonin gene fragment. Products of the expected DNA size wereidentified as ethidium bromide-stained DNA bands on agarose gels thatDNA was treated with T4 DNA polymerase and then purified from an agarosegel. Only the primer pair consisting of primers designated gelo-7 andgelo-5 yielded a relatively pure product of the expected size. Thesequences of degenerate primers gelo-7 and gelo-5 are set out belowusing IUPAC nucleotide symbols.

Gelo-7 (SEQ ID NO: 14) 5′ TTYAARGAYGCNCCNGAYGCNGCNTAYGARGG 3′

Gelo-5 (SEQ ID NO: 15) 3′ TTYTTYATRATRCANTGNCGNCANCTRGTYCA 5′

Primer gelo-7 corresponds to amino acids 87-97 of gelonin while primergelo-5 corresponds to amino acids 226-236. The blunt-ended DNA fragment(corresponding to amino acids 87 to 236 of gelonin) generated withprimers gelo-7 and gelo-5 was cloned into pUC18 (BRL, Gaithersburg,Md.). The DNA sequence of the insert was determined, and the deducedamino acid sequence based on the resulting DNA sequence matched theexperimentally determined gelonin amino acid sequence. The clonecontaining this gelonin segment was denoted pING3726.

The insert of clone pING3726 was labeled with ³²P and used as a probe toscreen the 150,000-member Gelonium cDNA library. Only one clonehybridized to the library plated in duplicate. This clone was purifiedfrom the library and its DNA sequence was determined. The clone containsa fragment encoding 185 of the 270 amino acids of gelonin (residues25-209) and is denoted pING3823.

2. Cloning Of The Fragment Encoding The N-Terminal Amino Acids OfGelonin

Based on the sequence determined for the gelonin gene segment inpING3726, exact oligonucleotide primers were designed as PCRamplification primers to be used in conjunction with a degenerate primerto amplify a 5′ gelonin gene fragment and with a nonspecific primer toamplify a 3′ gelonin gene fragment. cDNA generated using thePerkin-Elmer-Cetus RNA-PCR kit was amplified.

To amplify the 5′-end of the gelonin gene, PCR amplification with adegenerate primer gelo-1 and an exact primer gelo-10 was performed. Thesequences of the primers are set out below.

Gelo-1 (SEQ ID NO: 16) 5′ GGNYTNGAYACNGTNWSNTTYWSNACNAARGG 3′

Gelo-10 (SEQ ID NO: 17) 3′ TGTCTGAACCCGTAACTTGGTAA 5′

Primer gelo-1 corresponds to amino acids 1-11 of the gelonin gene whileprimer gelo-10 corresponds to amino acids 126-133. The product from thereaction was re-amplified with gelo-1 (SEQ ID NO: 16) and gelo-11 (anexact primer comprising sequences encoding amino acids 119-125 ofgelonin) to confer specificity to the reaction product. The sequence ofprimer gelo-11 is listed below.

Gelo-11 (SEQ ID No: 18) 3′ CACTCTTCCGTATATCTCTCTGT 5′

Hybridization with an internal probe confirmed that the desired specificgelonin DNA fragment was amplified. That fragment was cloned into pUC18and the vector generated was designated pING3727. The fragment wassequenced, revealing that the region of the fragment (the first 27nucleotides) corresponding to part of the degenerate primer gelo-1 couldnot be translated to yield the amino acid sequence upon which primergelo-1 was originally based. This was not unexpected considering thedegeneracy of the primer. The fragment was reamplified from the GeloniumcDNA with exact primers gelo-11 (SEQ ID NO: 18) and gelo-5′ (whichextends upstream of the 5′ end of the gelonin gene in addition toencoding the first 16 amino acids of gelonin). The sequence of primergelo-5′ is set out below.

Gelo-5′ (SEQ ID NO: 19) 5′ TCAACCCGGGCTAGATACCGTGTCATTCTCAACCAAAGGTGCCACTTATATTA 3′

The resulting DNA fragment encodes the first 125 amino acids of gelonin.While the majority of the sequence is identical to the natural geloningene, the first 32 nucleotides of the DNA fragment may be different. Forthe purposes of this application this N-terminal fragment is referred toas fragment GEL1-125.

3. Cloning Of The Fragment Encoding The C-Terminal Amino Acids OfGelonin

To amplify the 3′-end of the gelonin gene as well as 3′ untranslatedsequences, PCR amplification with exact primers gelo-9 and XE-dT wasperformed. The sequence of each of the primers is set out below.

Gelo-9 (SEQ ID NO: 20) 5′ CTTCATTTTGGCGGCACGTATCC 3′

XE-dT (SEQ ID NO: 21) 3′ TTTTTTTTTTTTTTTTTTTTTAG GGTGCATTCGAACGTCGGAGCTC5′

Primer gelo-9 corresponds to amino acids 107-113 of gelonin. PrimerXE-dT consists of a 3′ oligo-dT portion and a 5′ portion containing therestriction sites HindIII and XhoI, and will prime any poly A-containingcDNA. The reaction product was reamplified with exact primers gelo-8 andXE. The sequences of primers gelo-8 and XE are set out below.

Gelo-8 (SEQ ID NO: 22) 5′ CTCGCTGGAAGGTGAGAA 3′

XE (SEQ ID NO: 23) 3′ AGGGTGCATTCGAACGTCGGAGCTC 5′

Primer gelo-8 consists of sequences encoding amino acids 115-120 ofgelonin while the primer XE corresponds to the 5′ portion of the XE-dTprimer which contains HindIII and XhoI restriction sites. Hybridizationwith internal probes confirmed that the desired gelonin gene fragmentwas amplified. That fragment was then cloned into pUC18 by two differentmethods. First, it was cloned as a blunt-ended fragment into the SmaIsite of pUC18 (the resulting vector was designated pING3728) and,second, it was cloned as an EcoRI to HindIII fragment into pUC18 (thisvector was designated pING3729). Both vector inserts were sequenced. Theinsert of pING3728 encodes amino acids 114-270 of gelonin, while theinsert of pING3729 encodes amino acids 184-270 of gelonin plus other 3′sequences.

4. Assembly Of The Overlapping Gelonin DNA Fragments Into A CompositeGelonin Gene

To reassemble the C-terminal two-thirds of the gelonin gene, vectorpING3729 was cut with SspI (one SspI site is located within the vectorand the second is located about 80 bp downstream of the terminationcodon of the insert in the vector) and an XhoI linker (8 bp, New EnglandBiolabs) was ligated to the resulting free ends. The DNA was then cutwith XhoI and EcoRI, and the 350 bp fragment generated, encoding aminoacids 185-270 of gelonin, was isolated. This 350 bp fragment was ligatedadjacent to a NcoI to EcoRI fragment from pING3823 encoding amino acids37-185 of gelonin in a intermediate vector denoted pING3730, thusreassembling the terminal 87% of the gelonin gene (amino acids 37-270).

Next, fragment GEL1-125 was cut with SmaI and NcoI, resulting in afragment encoding amino acids 1-36 of gelonin which was ligated alongwith the NcoI to XhoI fragment of pING3730 into the vector pIC100.[pIC100 is identical to pING1500 described in Better, et al., Science,240:1041-1043 (1988) incorporated by reference herein], except that itlacks 37 bp upstream of the pelB leader sequence. The 37 bp wereeliminated by digestion of pING1500 with SphI and ECORI, treatment withT4 polymerase, and religation of the vector. This manipulationregenerated an EcoRI site in the vector while eliminating otherundesirable restriction sites.] Before ligation, the vector pIC100 hadpreviously been digested with SstI, treated with T4 polymerase, and cutwith XhoI. The ligation generated a new vector containing a completegelonin gene which was designated plasmid pING3731 and deposited withThe American Type Culture Collection, 12301 Parklawn Drive, Rockville,Md. 20852 on Oct. 2, 1991 as Accession No. 68721. The complete DNAsequence of the gelonin gene is set out in SEQ ID NO: 11.

EXAMPLE 2

A. Construction of Expression Vectors Containing the Gelonin Gene

A first E. coli expression vector was constructed containing the geloningene linked to the Erwinia carotovora pelB leader sequence, and to theSalmonella typhimurium araB promoter. A basic vector containing the arabpromoter is described in co-owned U.S. Pat. No. 5,028,530 issued Jul. 2,1991 which is incorporated by reference herein. The vector containingthe araB promoter was cut with EcoRI and XhoI. Two DNA fragments werethen ligated in tandem immediately downstream of the promoter. Thefragment ligated adjacent to the promoter was a 131 bp fragment derivedfrom SstI digestion, T4 polymerase treatment and digestion with EcoRI ofthe pIC100 vector which includes the leader sequence of the E.carotovora pe1B gene. The translated leader sequence is a signal forsecretion of the respective protein through the cytoplasmic membrane.The fragment ligated downstream of the leader sequence was a SmaI toXhoI fragment from pING3731 which contains the complete gelonin gene.Thus, the expression vector contains the gelonin gene linked to the pelBleader sequence and the arab promoter. This plasmid is designatedpING3733.

A second expression vector may be constructed that is identical to thefirst except that the gelonin gene sequences encoding the nineteenC-terminal amino acids of gelonin are not included. The cDNA sequence ofthe gelonin gene predicted a 19 residue C-terminal segment that was notdetected in any peptide fragments generated for determination of thegelonin amino acid sequence. These 19 amino acids may represent apeptide segment that is cleaved from the mature toxinpost-translationally, i.e. that is not present in the native protein. Asimilar C-terminal amino acid segment was identified in the plant toxinα-trichosanthin [Chow et al., J. Biol. Chem., 265:8670-8674 (1990)].Therefore, the expression product without the C-terminal fragment is ofinterest.

For construction of a gelonin expression vector without the 19C-terminal amino acids of gelonin, PCR was used to amplify and alter the3′-end of the gene. pING3728 was amplified with primers gelo-14 andgelo-9 (SEQ ID NO: 20). The sequence of primer gelo-14 is set out below.

Gelo-14 (SEQ ID NO: 24) 5′ TGATCTCGAGTACTATTTAGGATCTTTATCGACGA 3′

Primer gelo-14, which corresponds to gelonin amino acids 245-256,introduces a termination codon (underlined in the primer sequence) inthe gelonin gene sequence which stops transcription of the gene beforethe sequences encoding the terminal 19 amino acids of the gelonin andalso introduces a XhoI site immediately downstream of the terminationcodon. The PCR product was cut with XhoI and EcoRI, and the resulting208 bp fragment encoding amino acids 185-251 of gelonin was purifiedfrom an agarose gel. This fragment was ligated adjacent to the NcoI toEcoRI fragment from pING3823 encoding amino acids 37-185 of gelonin togenerate plasmid pING3732. A final expression vector, pING3734,containing a gelonin gene with an altered 3′-end was generated bysubstituting an NcoI to XhoI fragment encoding amino acids 37-251 ofgelonin from pING3732 into pING3733.

B. Identification of the Native Gelonin 5′-End

Inverse PCR was used to identify a cDNA clone encoding the 5′-end of themature gelonin gene. 5 μg of total G. multiflorum RNA was converted tocDNA using the Superscript Plasmid System (BRL, Gaithersburg, Md.) withGelo-11 (SEQ ID NO: 18) as a primer. Gelonin cDNA was self-ligated togenerate covalent circular DNA and the ligated DNA was amplified by PCRwith oligonucleotides Gelo-9 (SEQ ID NO: 20) and Gelo-16. The sequenceof primer Gelo-16 is set out below.

Gelo-16 (SEQ ID NO: 25) 5′ GTAAGCAGCATCTGGAGCATCT 3′

The PCR product was size-fractionated on an agarose gel and DNAs largerthan 300 bp were cloned into SmaI cut pUC18. Several clones weresequenced with the primer Gelo-18, the sequence of which is set outbelow.

Gelo-18 (SEQ ID NO: 26) 5′ CATTCAAGAAATTCACGTAGG 3′

A clone identified as having the largest gelonin-specific insert wasdesignated pING3826. The DNA sequence of pING3826 included the first 32nucleotides of the natural, mature gelonin gene not necessarily presentin gelonin expression plasmids pING3733 and pING3734. The complete DNAsequence of the natural gelonin gene is set out in SEQ ID NO: 11.

C. Construction of Expression Vectors Containing a Gelonin Gene with aNatural 5′ End

Derivatives of expression vectors pING3733 and pING3734 (describedabove) containing a gelonin gene with the natural 5′ sequence weregenerated as follows. The 5′-end of gelonin was amplified from pING3826with the PCR primers Gelo-16 (SEQ ID NO: 24) and Gelo-17, the sequenceof which is set out below.

Gelo-17 (SEQ ID NO: 27) 5′ GGCCTGGACACCGTGAGCTTTAG 3′

The 285 bp PCR product was treated with T4 polymerase and cut with NcoI.The resulting 100 bp 5′-end DNA fragment was isolated from an agarosegel and ligated adjacent to the 120 bp pelB leader fragment from plC100(cut with SstI, treated with T4 polymerase and cut with PstI) intoeither pING3733 or pING3734 digested with PstI and NcoI. The resultingplasmids pING3824 and pING3825 contain the entire native gelonin geneand the native gelonin gene minus the nineteen amino acid carboxylextension, respectively, linked to the pelB leader and under thetranscriptional control of the araB promoter. The gene construct withoutthe nineteen amino acid carboxyl extension in both pING3734 and pING3825encodes a protein product referred to in this application as“recombinant gelonin”.

D. Purification of Recombinant Gelonin

Recombinant gelonin was purified by the following procedure: E. colifermentation broth was concentrated and buffer-exchanged to 10 mM sodiumphosphate at pH 7.0 by using an S10Y10 cartridge over a DC10 unit(Amicon) the concentrated and buffer-exchanged material was applied to aCM52 column (100 g, 5×10 cm). The column was washed with 1 L of startingbuffer and eluted with a 0 to 300 mM NaCl gradient in starting buffer(750 ml total volume). The pure gelonin containing fractions were pooled(elution was from 100-250 mM NaCl), concentrated over an Amicon YM10membrane, equilibrated with 10 mM sodium phosphate buffer, pH 7.0, andstored frozen at −20° C. A further purification step was attempted usingBlue Toyopearl chromatography. However, this procedure did not result inan increased purity of material and resulted in an approximate 50% lossof the starting material.

EXAMPLE 3

Assembly of Gelonin Genes with Cysteine Residues Available forConjugation

The wild-type gelonin protein has two cysteine residues at positions 44and 50 which are linked by an endogenous disulfide bond. The proteincontains no free cysteine residue directly available for conjugation toantibodies or other proteins. Analogs of gelonin which contain a freecysteine residue available for conjugation were generated by threedifferent approaches. In one approach, various residues along theprimary sequence of the gelonin were replaced with a cysteine residue,creating a series of analogs which contain an odd number of cysteineresidues. In another approach, one of the two endogenous cysteines wasreplaced by alanine, creating a molecule which lacks an intrachaindisulfide bond but contains a single, unpaired cysteine. In yet anotherapproach both endogenous cysteines were replaced by alanines and a thirdnon-cysteine residue was replaced by a cysteine, creating an analog witha single, unpaired cysteine.

Fifteen analogs of gelonin were constructed. Ten non-cysteine residuesof gelonin were targeted for substitution with a cysteine residue.Comparison of the amino acid sequence of gelonin to the natural aminoacid sequence and tertiary structure of the ricin A-chain (see FIG. 1)suggested that these positions would be at the surface of the moleculeand available for conjugation. Each of the ten gelonin analogs include acysteine substituted in place of one of the following residues:lysine₁₀, asparagine₆₀, isoleucine₁₀₃, aspartic acid₁₄₆, arginine₁₈₄,serine₂₁₅, asparagine₂₃₉, lysine₂₄₄, aspartic acid₂₄₇, and lysine₂₄₈,and the analogs have respectively been designated Gel_(C10), Gel_(C60),Gel_(C103), Gel_(C146), Gel_(C184), Gel_(C215), Gel_(C239), Gel_(C244),Gel_(C247), and Gel₂₄₈.

Two analogs of gelonin were constructed in which one of the nativegelonin cysteines that participates in an endogenous disulfide bond wasreplaced with a non-cysteine residue. Specifically, the cysteine atposition 50 was replaced with an alanine residue, creating a geloninanalog (designated Gel_(A50(C44)), shown in SEQ ID NO: 99) which has acysteine available for disulfide bonding at position 44. TheGel_(A50(C44)) analog has been referred to previously as Gel_(C44) (see,e.g., co-owned, co-pending U.S. patent application Ser. No. 07/988,430,incorporated by reference herein). Conversely, the cysteine at position44 was replaced with an alanine residue, resulting in an analog(designated Gel_(A44(C50)), shown in SEQ ID NO: 100) which has acysteine available for disulfide bonding at position 50. TheGel_(A44(C50)) analog has been referred to previously as Gel_(C50) (see,e.g., co-owned, co-pending U.S. patent application Ser. No. 07/988,430,incorporated by reference herein). The combined series of the foregoingtwelve analogs thus spans the entire length of the mature geloninprotein.

Another gelonin analog (Gel_(A44A50) SEQ ID NO: 101) was constructed inwhich both native gelonin cysteines were replaced with alanines. TheGel_(A44A50) analog has been referred to previously as Gel_(C44AC30A)(see, e.g., co-owned, co-pending U.S. patent application Ser. No.07/988,430, incorporated by: reference herein). Two additional analogswere constructed which have alanine residues substituted in place ofboth native cysteines and have either a cysteine residue substituted inplace of the native lysine at position 10 (Gel_(C10A44A50), shown in SEQID NO: 110) or a cysteine residue substituted in place of the nativeaspartate at position 247 (Gel_(C247A44A50), shown in SEQ ID NO: 111).

The variants of recombinant gelonin were constructed by restrictionfragment manipulation or by overlap extension PCR with syntheticoligonucleotides. The sequences of the primers used for PCR are set outbelow. In each mutagenic primer sequence, the nucleotides correspondingto the changed amino acid, either a cysteine or an alanine residue, areunderlined.

Gelo-9 (SEQ ID NO: 20)

Gelo-11 (SEQ ID NO: 18)

Gelo-16 (SEQ ID NO: 25)

Gelo-17 (SEQ ID NO: 27)

Gelo-18 (SEQ ID NO: 26)

Gelo-19 (SEQ ID NO: 58) 5′ CAGCCATGGAATCCCATTGCTG 3′

GeloC-1 (SEQ ID NO: 28) 5′ TCGATTGCGATCCTAAATAGTACTC 3′

GeloC-2 (SEQ ID NO: 29) 5′ TTTAGGATCGCAATCGACGAACTTCAAG 3′

GeloC-3-2 (SEQ ID NO: 30) 5′ GTTCGTCTGTAAAGATCCTAAATAGTACTCGA 3′

GeloC-4 (SEQ ID NO: 31) 5′ GGATCTTTACAGACGAACTTCAAGAGT 3′

GeloC-5 (SEQ ID NO: 32) 5′ TCTTGTGCTTCGTCGATAAAGATCC 3′

GeloC-6 (SEQ ID NO: 33) 5′ ATCGACGAAGCACAAGAGTGCTATTTT 3′

GeloC-9 (SEQ ID NO: 34) 5′ GTAAAACCATGCATAGCACTCTTGAAGTTCGT 3′

GeloC-10 (SEQ ID NO: 35) 5′ AGTGCTATGCATGGTTTTACTTGATCAACTGC 3′

GeloC-13 (SEQ ID NO: 36) 5′ AGCACATGTGGTGCCACTTATATTACCTA 3′

GeloC-14 (SEQ ID NO: 37) 5′ TAAGTGGCACCACATGTGCTAAAGCTCACGGTG 3′

GeloC-15 (SEQ ID NO: 38) 5′ TGACTGTGGACAGTTGGCGGAAATA 3′

GeloC-16 (SEQ ID NO: 39) 5′ GCCAACTGTCCACAGTCATTTGAAAGCGCTACC 3′

GeloC-17 (SEQ ID NO: 40) 5′ GATGATCCTGGAAAGGCTTTCGTTTTGGTAGCGCTT3′

GeloC-18 (SEQ ID NO: 41) 5′ AAGCCTTTCCAGGATCATCAGCTTTTTTGCGCAGCAATGGG 3′

GeloC-19 (SEQ ID NO: 42) 5′ AAGCCTTTCCAGGATCATCACAT 3′

GeloC-20 (SEQ ID NO: 59) 5′ CACATGTAAAACAAGACTTCATTTTGGC 3′

GeloC-21 (SEQ ID NO: 60) 5′ TGAAGTCTTGTTTTAGATGTGTTTTTGAAGAGGCCT 3′

GeloC-22 (SEQ ID NO: 61) 5′ ATGCCATATGCAATTATAAACCAACGGAGA 3′

GeloC-23 (SEQ ID NO: 62) 5′ GGTTTATAATTGCATATGG CATTTTCATCAAGTTTCTTG 3′

GeloC-24 (SEQ ID NO: 63) 5′ CTTTCAACAATGCATTCGCCCGGCGAATAATAC 3′

GeloC-25 (SEQ ID NO: 64) 5′ GCGAATGCATTGTTGAAAGTTATTTCTAATTTG 3′

GeloC-26 (SEQ ID NO: 65) 5′ GTTTTGTGAGGCAGTTGAATTGGAAC 3′

GeloC-27 (SEQ ID NO: 66) 5′ TTCAACTGCCTCACAAAACATTCCATTTGCACCT 3′

GeloC-28 (SEQ ID NO: 67) 5′ AAAAGCTGATGATCCTGGAAAGTG 3′

GeloC-29 (SEQ ID NO: 68) 5′ TCCAGGATCATCAGCTTTTTTGCGCAGCAATGGGA 3′

araB2 (SEQ ID NO: 43) 5′ GCGACTCTCTACTGTTTC 3′

HINDIII-2 (SEQ ID NO: 44) 5′ CGTTAGCAATTTAACTGTGAT 3′

(1) Specifically, a cysteine was introduced at amino acid 247 of gelonin(which is normally occupied by an aspartic acid which corresponds to thecysteine at position 259 in the ricin A-chain) by PCR with mutagenicprimers GeloC-3-2 and GeloC-4 in conjunction with primers HINDIII-2 (aprimer located in the vector portion of pING3734 or pING3825), Gelo-9and Gelo-8. Template DNA (pING3734) was amplified with GeloC-3-2 andHINDIII-2 and in a concurrent reaction with GeloC-4 and Gelo-9. Theproducts of these reactions were mixed and amplified with the outsideprimers Gelo-8 and HINDIII-2. The reaction product was cut with EcoRIand XhoI, purified, and was inserted into plasmid pING3825 in athree-piece ligation. The DNA sequence of the Gel_(C247) variant (SEQ IDNO: 102) was then verified. The plasmid containing the sequence encodingGel_(C247) was designated pING3737 and was deposited with the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 onJun. 9, 1992 as ATCC Accession No. 69009.

(2-3) In the same manner, a cysteine residue was introduced in place ofthe amino acid at position 248 (a lysine) of gelonin with the mutagenicoligonucleotides GeloC-1 and GeloC-2 to generate analog Gel_(C248) (SEQID NO: 103) in plasmid pING3741, and a cysteine residue was introducedat amino acid position 239 (normally occupied by a lysine) with primersGeloC-9 and GeloC-10 to generate analog Gel₂₃₉ (SEQ ID NO: 104) inplasmid pING3744.

(4) Also in the same manner, a cysteine residue was introduced at aminoacid 244 (a lysine) of gelonin with mutagenic primers GeloC-5 andGeloC-6 to generate analog Gel_(C244) (SEQ ID NO: 105) in a plasmiddesignated pING3736. This variant was prepared by PCR using plasmidpING3734 as template DNA rather than pING3825. It therefore encodes thesame N-terminal gelonin amino acid sequence as plasmids pING3737,pING3741, and pING3744, but includes the PCR primer-derived 5′-end 32nucleotides instead of the native gelonin 5′-end nucleotides.

(5) A cysteine residue was introduced in place of the amino acid(normally occupied by a lysine) at position 10 of gelonin by a similarprocedure. A cysteine was introduced with mutagenic primers GeloC-13 andGeloC-14 by amplifying pING3824 with araB2 (a vector primer) andGeloC-14, and in a separate reaction, with GeloC-13 and Gelo-11. Thesereaction products were mixed and amplified with the outside primersaraB2 and Gelo-11. The PCR product was cut with PstI and NcoI, purified,and cloned back into pING3825 to generate analog Gel_(C10) (SEQ ID NO:106) in the plasmid designated pING3746 and deposited with the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 onJun. 9, 1992 as ATCC Accession No. 69008.

(6) The asparagine at position 60 of gelonin was replaced with acysteine residue using two mutagenic oligos, GeloC-15 and GeloC-16, inconjunction with oligos araB2 and Gelo-11 in the same manner as for theGel_(C10) variant. The plasmid encoding the Gel_(C60) (SEQ ID NO: 107)analog was designated pING3749.

(7) A cysteine was introduced at amino acid 103 (an isoleucine) by PCRwith mutagenic primers GeloC-20 and GeloC-21 in conjunction with primersaraB2 and HINDIII-2. Template DNA (pING3733) was amplified with GeloC-21and araB2 and separately with GeloC-20 and HINDIII-2. The products ofthese reactions were mixed and amplified with the outside primers araB2and HINDIII-2. The reaction product was cut with NcoI and BclI,purified, and inserted into pING3825 digested with NcoI and BclI. Theoligonucleotides used to place a cysteine at residue 103 also introducedan AflIII restriction site which was verified in the cloned gene. Theplasmid containing the Gel_(C103) (SEQ ID NO: 108) analog was designatedpING3760.

(8) A cysteine was introduced at position 146 (an aspartic acid) by asimilar strategy. Template DNA (pING3733) was amplified with mutagenicprimer GeloC-22 and Gelo-14 and separately with mutagenic primerGeloC-23 and Gelo-19. The products of these reactions were mixed, andamplified with Gelo-19 and Gelo-14. The reaction product was cut withBglII and EcoRI, and can be inserted into pING3825 in a three-pieceligation. The oligonucleotides used to place a cysteine at residue 146also introduced a NdeI restriction site which can be verified in thecloned gene.

(9) To introduce a cysteine at position 184 (normally occupied by anarginine) of gelonin, template DNA (pING3733) was amplified withmutagenic primer GeloC-25 and araB-2 and separately with mutagenicprimer GeloC-24 and HINDIII-2. The products of these reactions weremixed, and amplified with araB2 and Gelo-14. The reaction product wascut with NcoI and BclI, and inserted into pING3825 previously digestedwith NcoI and BclI. The oligonucleotides used to place a cysteine atresidue 184 also introduced an NsiI restriction site which was verifiedin the cloned gene. The plasmid containing the sequence encoding theGel_(C184) (SEQ ID NO: 109) variant was designated pING3761.

(10) A cysteine may be introduced at position 215 (a serine) by asimilar strategy. Template DNA (pING3733) was amplified with mutagenicprimer GeloC-27 and araB2 and separately with mutagenic primer GeloC-26and HINDIII-2. The products of these reactions were mixed, and amplifiedwith araB2 and HINDIII-2. The reaction product was cut with EcoRI andBclI, and may be inserted into pING3825 in a three-piece ligation.

(11) Another gelonin variant with a free cysteine residue was generatedby replacing on* of the two naturally occurring gelonin cysteineresidues, the cysteine a position 50, with an alanine. Plasmid pING3824was amplified with primers GeloC-17 and Gelo-11, and concurrently in aseparate reaction with primers GeloC-19 and araB2. The reaction productswere mixed and amplified with araB2 and Gelo-11. This product was cutwith NcoI and BglII, and cloned back into the vector portion of pING3825to generate pING3747 (ATCC 69101). This analog was designatedGel_(A50(C44)) and it contains a cysteine available for disulfidebonding at amino acid position 44. Non-cysteine residues, other thanalanine, which do not disrupt the activity of gelonin, also may beinserted at position 50 in natural gelonin in order to generate agelonin analog with a single cysteine at position 44.

(12) A gelonin variant in which the natural cysteine at position 44 waschanged to alanine was constructed by amplifying pING3733 using themutagenic oligonucleotides GeloC-28 and GeloC-29 in conjunction withprimers araB2 and HINDIII-2. The amplified DNA was cut with NcoI andBglII and cloned into a gelonin vector, generating pING3756. Thatvariant generated was designated Gel_(A44(C50)). Non-cysteine residues,other than alanine, which do not disrupt gelonin activity, also may beinserted at position 44 in order to generate a gelonin analog with asingle cysteine at position 50.

(13) A gelonin variant in which both the cysteine at position 44 and thecysteine at position 50 of gelonin were changed to alanine residues wasconstructed by overlap PCR of pING3824 using the mutagenicoligonucleotides GeloC-17 and GeloC-18 in conjunction with primers araB2and Gelo-11. This analog, like the native gelonin protein, has nocysteine residues available for conjugation. The plasmid encoding theanalog was designated pING3750. The analog generated was designatedGel_(A44A50) (SEQ ID NO: 101). Non-cysteine residues, other thanalanine, which do not disrupt gelonin activity, also may be substitutedat both positions 44 and 50 in order to generate a gelonin analog withno cysteine residues.

(14) The triple mutant Gelonin_(C247A44A50) (SEQ ID NO: 111) wasconstructed from the plasmids pING3824, pING3750 and pING3737. Thisvariant contains an introduced cysteine at position 247 while both ofthe naturally occurring cysteine residues at positions 44 and 50 havebeen replaced with alanine. The analog is desirable because, in thisanalog, disulfide linkage to an antibody is only assured at a singlecysteine residue. Plasmid pING3824 was cut with NcoI and XhoI and thevector fragment was purified in an agarose gel. pING3750 was cut withNcoI and EcoRI and pING3737 was cut with EcoRI and XhoI. The NcoI-EcoRIfragment encodes the alanines at positions 44 and 50 while theEcoRI-XhoI fragment encodes the cysteine at position 247. Each of thesefragments was purified and ligated to the NcoI to XhoI vector fragment.The resulting plasmid is named pING3752.

(15) The triple mutant Gelonin_(C10A44A50) (SEQ ID NO: 110) was alsoconstructed by assembly from previously assembled plasmids. In thiscase, pING3746 was cut with PstI and NcoI, while pING3750 was cut withNcoI and XhoI. Each of the insert fragments were purified byelectrophoresis in an agarose gel, and the fragments were ligated into aPstI and XhoI digested vector fragment. The resulting vector wasdesignated pING3753. The Gel_(C10A44A50) analog has been referred topreviously as Gel_(C10C44AC50A) (see, e.g., co-owned, co-pending U.S.patent application Ser. No. 07/988,430, incorporated by referenceherein).

Each of the gelonin variants constructed was transformed into E. colistrain E104. Upon induction of bacterial cultures with arabinose,gelonin polypeptide could be detected in the culture supernatants withgelonin-specific antibodies. There were no significant differencesdetected in the expression levels of gelonin from plasmids pING3734 andpING3825, or in the levels from any of the gelonin variants. Eachprotein was produced in E. coli at levels of approximately 1 g/l.

EXAMPLE 4

Reticulocyte Lysate Assay

The ability of gelonin and recombinant gelonin analogs to inhibitprotein synthesis in vitro was tested using a reticulocyte lysate assay(RLA) described in Press et al., Immunol. Letters, 14:37-41 (1986). Theassay measures the inhibition of protein synthesis in a cell-free systemusing endogenous globin mRNA from a rabbit red blood cell lysate.Decreased incorporation of tritiated leucine (³H-Leu) was measured as afunction of toxin concentration. Serial log dilutions of standard toxin(the 30 kD form of ricin A-chain, abbreviated as RTA 30), nativegelonin, recombinant gelonin (rGelonin or rGel) and gelonin analogs weretested over a range of 1 μg/ml to 1 pg/ml. Samples were tested intriplicate, prepared on ice, incubated for 30 minutes at 37° C., andthen counted on an Inotec Trace 96 cascade ionization counter. Bycomparison with an uninhibited sample, the picomolar concentration oftoxin (pM) which corresponds to 50% inhibition of protein synthesis(IC₅₀) was calculated. As is shown in Table 1 below, recombinant geloninand most of its analogs exhibit activity in the RLA comparable to thatof native gelonin. For some of the analogs (such as Gel_(C239)), RLAactivity was diminished.

TABLE 1 Toxin IC₅₀ (pM) RTA 30 2.5 Gelonin 15 rGelonin 11 Gel_(C10) 60Gel_(A50(C44)) 20 Gel_(A44(C50)) 47 Gel_(C60) 26 Gel_(C239) 955Gel_(C244) 32 Gel_(C247) 12 Gel_(C248) 47 Gel_(A44A50) 16Gel_(C10A4450A) 7 Gel_(C247A44A50) 20

EXAMPLE 5

Human-Engineered Antibodies for Construction of Immunotoxins

Antibodies for use in constructing immunotoxins according to the presentinvention may be humanized antibodies, such as he3 and fragments thereofwhich display increased content of human amino acids and a high affinityfor human CD5 cell differentiation marker. he3 is a humanized form of amouse H65 antibody (H65 is a preferred monoclonal antibody for use inpreparing humanized antibodies according to the present invention and isproduced by hybridoma cell line XMMLY-H65 (H65) deposited with theAmerican Type Culture Collection in Rockville, Md. (A.T.C.C.) and giventhe Accession No. HB9286).

Humanized antibodies for use in the present invention are prepared asdisclosed herein using the humanized forms of the murine H65 antibody inwhich both low and moderate risk changes described below were made inboth variable regions. Such humanized antibodies should have lessimmunogenicity and have therapeutic utility in the treatment ofautoimmune diseases in humans. For example, because of their increasedaffinity over existing therapeutic monoclonal antibodies such as H65,he3 antibodies of the invention may be administered in lower doses thanH65 anti-CD5 antibodies in order to obtain the same therapeutic effect.

Humanized antibodies, such as he3, are useful in reducing theimmunogenicity of foreign antibodies and also results in increasedpotency when used as a portion of an immunoconjugate.

Construction of humanized antibody variable domains according to thepresent invention and for use as components of immunotoxins may be basedon a method which includes the steps of: (1) identification of the aminoacid residues of an antibody variable domain which may be modifiedwithout diminishing the native affinity of the domain for antigen whilereducing its immunogenicity with respect to a heterologous species; and(2) the preparation of antibody variable domains having modifications atthe identified residues which are useful for administration toheterologous species. The methods of the invention are based on a modelof the antibody variable domain described herein and in U.S. patentapplication Ser. No. 07/808,464 by Studnicka, et al., which predicts theinvolvement of each amino acid in the structure of the domain.

Unlike other methods for humanization of antibodies, which advocatereplacement of the entire classical antibody framework regions withthose from a human antibody, the methods described herein and in U.S.patent application Ser. No. 07/808,464 by Studnicka, et al., nowabandoned, introduce human residues into the variable domain of anantibody only in positions which are not critical for antigen-bindingactivity and which are likely to be exposed toimmunogenicity-stimulating factors. The present methods are designed toretain sufficient natural internal structure of the variable domain sothat the antigen-binding capacity of the modified domain is notdiminished in comparison to the natural domain.

The human consensus sequences in which moderate risk residues areconverted from mouse residues to human residues are represented in FIGS.10A and 10B as lines labelled hK1 (i.e., subgroup 1 of the human kappachain) and hH3 (i.e., subgroup 3 of the human heavy chain). Symbols inthe figures for conservation and for risk in “bind” and “bury” lines areas follows:

First Symbol in Pair (Ligand Binding)

+ Little or not direct influence on antigen-binding loops, low risk ifsubstituted

∘ Indirectly involved in antigen-binding loop structure, moderate riskif changed

− Directly involved in antigen-binding loop conformation or antigencontact, great risk if modified

Second Symbol in Pair (Immunogenicity/Struture)

+ Highly accessible to solvent, high immunogenicity, low risk ifsubstituted

∘ Partially buried, moderate immunogenicity, moderate risk if altered

− Completely buried in subunit's hydrophobic core, low immunogenicity,high risk if changed

= Completely buried in interface between subunits, low immunogenicity,high risk if modified

Significance of Pairs

++ Low risk

Highly accessible to solvent and high immunogenicity, but little or noeffect on specific antigen binding

∘+, +∘, ∘∘ Moderate Risk

Slight immunogenicity or indirect involvment with antigen binding

any − or = High risk

Buried within the subunit core/interface or strongly involved in antigenbinding, but little immunogenic potential

In the line labelled “mod”, a dot (.) represents a residue which may bemutated from “mouse” to “human” at moderate risk. There are 29 suchmoderate risk positions.

The mouse residue matches the human consensus residue more than 50% ofthe time at 131 positions (102 positions match 90%-100% and 29 positionsmatch 50% to 90%). These positions were not changed.

The lines labelled M/H in FIGS. 12A and 12B indicate the 91 positionswhich differed significantly between the mouse and human sequences(i.e., where the human sequences have the mouse residue less than 50% ofthe time). Moderate risk positions, designated m in the M/H line, werekept “mouse”; whereas those designated H or h were changed to human. The25 low risk positions which were already human-like or which werepreviously humanized (as described supra in Example 2) are designated“{circumflex over ( )}” in the M/H line. Finally, the 54 high riskpositions in which the mouse and human residues did not match aredesignated M and are kept “mouse”.

Fifteen differences occur at moderate risk positions at which the mouseand human sequences differ. At ten of those positions (designated “H” onthe M/H lines of FIG. 6) the mouse residue aligns with a human consensusamino acid which is highly conserved. Therefore, the mouse residue atthat position is identified as one to be changed to the conservedhuman-residue.

At moderate risk positions (designated “m”) in which the mouse and thehuman sequences differ, the mouse residue aligns with a human consensusamino acid which is moderately conserved. However, since the mouseresidue is found at that position in other actual sequences of humanantibodies [See Kabat, et al., sequences of Proteins of ImmunoglobulinInterest,Fourth Edition, U.S. Department of Health and Human Services,Public Health Service, National Institutes of Health (1987)] thepositions are identified as ones to be kept “mouse.” Although there areno such positions in this particular sequence, such positions may occurin other antibodies.

At four moderate risk positions (designated “h”), the mouse residuealigns with a human consensus amino acid which is moderately conservedbut the mouse residue is not found at that position in an actual humanantibody sequence in Kabat, et al. Sequences of Proteins ofImmunoglobulin Interest, supra. Therefore, that position is identifiedas ones to be changed to “human.”

At one moderate risk position (designated “m”) in which the mouse andhuman sequences differ, the mouse residue aligns with a human consensusamino acid which is poorly conserved. Therefore, that position isidentified as one to be kept “mouse.”

A. Assembly of Moderate Risk Heavy Chain Expression Vectors

The humanized H65 heavy chain containing the moderate risk residues wasassembled by the following strategy. The moderate-risk expression vectorwas assembled from intermediate vectors. The six oligonucleotidesequences (oligos), disclosed in FIG. 12 and labelled HUH-G11, HUH-G12,HUH-G3, HUH-G4, HUH-G5, and HUH-G6 (the sequences of HUH-G11 and HUH-G12are set out in SEQ ID Nos. 131 and 132 and HUH-G3, HUH-G4, HUH-G5, andHUH-G6 are set out in SEQ ID NOS: 137-140) were assembled by PCR.Oligonucleotides containing the synthetic humanized antibody gene weremixed in pairs (HUH-G11+HUH-G12, HUH-G3+HUH-G4, and HUH-G5+HUH-G6) in a100 μl reaction with 1 μg of each DNA and filled in as described above.A portion of each reaction product was mixed in pairs (HUH-G11,12+HUH-G3, 4; HUH-G3, 4+HUH-G5, 6), 2.5 U Taq was added and samples werereincubated as described above. The V-J region was assembled by mixingequal amounts of the HUH-G11, 12, 3, 4 reaction product with the HUH-G3,4, 5, 6 product, followed by PCR with 0.5 ug of primers H65G-2S andH65-G2 as described above. The reaction product was cut with SalI andBstEII and cloned into the expression vector, similar to that describedfor heavy chain in Robinson et al., Hum. Antibod. Hybridomas 2:84(1991), generating pING4617. That plasmid was sequenced with Sequenase(USB, Cleveland), revealing that two residues were altered (a G-A atposition 288 and a A-T at position 312, numbered from the beginning ofthe leader sequence). The correct variable region was restored bysubstitution of this region from pING4612, generating the expectedV-region sequence in pING4619.

An intermediate vector containing the other moderate-risk changes wasconstructed by PCR assembly of the oligos HUH-G13, HUH-G14, HUH-G15, andHUH-G16 (FIG. 11 and SEQ ID Nos: 133-136). Oligos HUH-G13+HUH-G14 andHUH-G15+HUH-G16 were mixed-and filled in with Vent polymerase (NewEngland Biotabs) in a reaction containing 10 mM KCl, 20 mM TRIS pH 8.8,10 mM (NH₄)₂SO₂, 2 mM MgSO₄, 0.1% Triton X-100, 100 ng/ml BSA, 200 uM ofeach dNTP, and 2 units of Vent polymerase in a total volume of 100 μl.The reaction mix was incubated at 94° C. for 1 minute, followed by 2minutes at 50° C. and 20 minutes at 72° C. The reaction products (40 μl)were mixed and amplified with the oligonucleotides H65-G13 and H65-G2with Vent polymerase in the same reaction buffer and amplified for 25cycles with denaturation at 94° C. for 1 minute, annealing at 50° C. for2 minutes and polymerization at 72° C. for 3 minutes. The reactionproduct was treated with T4 polymerase and then digested with AccI. The274 base pair (bp) fragment was purified on an agarose gel and ligatedalong with the 141 bp SalI to AccI fragment from pING4619 into pUC18 cutwith SalI and SmaI to generate pING4620. pING4620 contains the entiresignal sequence, V-region, and J-region of the moderate-risk H65 heavychain.

The final expression vector for the moderate-risk H65 heavy chain,pING4621, was assembled by cloning the SalI to BstEII fragment frompING4620 into the same expression vector described above.

B. Assembly of Moderate-Risk Light Chain Expression Vectors

The moderate-risk humanized V- and J-segments of the light chain wereassembled from six oligonucleotides, $H65K-1 (SEQ ID NO: 117), HUH-K7(SEQ ID NO: 119), HUH-K6 (SEQ ID NO: 118), HUH-K8 (SEQ ID NO: 120),HUH-K4 (SEQ ID NO: 121 and HUH-K5 (SEQ ID NO: 122). The oligonucleotideswere amplified with PCR primers H65K-2S and JK1-HindIII.Oligonucleotides containing the synthetic humanized antibody gene weremixed in pairs ($H65-K1+HUH-K7, HUH-K6+HUH-K4+HUH-K5) and incubated withVent polymerase as described for the moderate-risk heavy chain. Aportion of each reaction product (40 ul) was mixed in pairs($H65H-K1/HUH-K7+HUH-K6, 8; HUH-K6, 8+HUH-K4, 5) and filled in as above.The light chain gene was then assembled by amplifying the full lengthgene with the PCR primers H65K-2S and JK1-HindIII with Vent polymerasefor 25 cycles as outlined above. The assembled V/J region was cut withSalI and HindIII, purified by electrophoresis on an agarose gel, andassembled into a light chain antibody expression vector, pING4630.

EXAMPLE 6

Transfection of he3 Genes and Purification of Expression Products

A. Stable Transfection of Mouse Lymphoid Cells for the Production of he3Antibody

The cell line Sp2/0 (American Type culture Collection Accession No.CRL1581) was grown in Dulbecco's Modified Eagle Medium plus 4.5 g/lglucose (DMEM, Gibco) plus 10% fetal bovine serum. Media weresupplemented with glutamine/penicillin/streptomycin (Irvine Scientific,Irvine, Calif.).

The electroporation method of Potter, H., et al., Proc. Natl. Acad.Sci., USA, 81:7161 (1984) was used. After transfection, calls wereallowed to recover in complete DMEM for 24-48 hours, and then seeded at10,000 to 50,000 calls per well in 96-well culture plates in thepresence of selective medium. Histidinol (Sigma) selection was at 1.71μg/ml, and mycophenolic acid (Calbiochem) was at 6 μg/ml plus 0.25 mg/mlxanthine (Sigma). The electroporation technique gave a transfectionfrequency of 1-10×10⁻⁵ for the Sp2/0 cells.

The he3 light chain expression plasmid pING4630 was linearized bydigestion with PvuI restriction endonuclease and transfected into Sp2/0cells, giving mycophenolic acid—resistant clones which were screened forlight chain synthesis.

Four of the top-producing subclones, secreting 4.9-7.5 μg/ml werecombined into two pools (2 clones/pool) and each pool was transfectedwith plasmid pING42621, containing the moderate-risk heavy chain. Afterselection with histidinol, the clones producing the most light plusheavy chain, Sp2/0-4630 and 4621 Clones C1705 and C1718, secretedantibody at approximately 15 and 22 μg/ul respectively in the presenceof 10⁻⁷ M dexamethasone in an overgrown culture. in a T25 flask. CloneC1718 was deposited with the American Type Culture Collection, 1230Parklawn Drive, Rockville, Md., 20852 on Dec. 1, 1992 as ATCC HB 11206.The best producer is a subclone of Clone C1718 which is produced bylimiting dilution subcloning of Clone C1718.

B. Purification of he3 Antibody Secreted in Tissue Culture

Sp2/0-4630+4621 Clone C1705cells were grown in culture medium HB101(Hana Biologics)+1% Fetal Bovine Serum, supplemented with 10 mM HEPES,1×Glutamine-Pen-Strep (Irvine Scientific #9316). The spent medium wascentrifuged at about 5,000×g for 20 minutes. The antibody level wasmeasured by ELISA. Approximately 200 ml of cell culture supernatant wasloaded onto a 2 ml Protein A-column (Sigma Chemicals), equilibrated withPBS (buffer 0.15 M NaCl, 5 mM sodium phosphate, 1 mM potassiumphosphate, buffer pH 7.2). The he3 antibody was eluted with a step pHgradient (pH 5.5, 4.5 and 2.5). A fraction containing he3 antibody (9%yield) but not bovine antibody, was neutralized with 1 M Tris pH 8.5,and then concentrated 10-fold by Centricon 30 (Amicon) diluted 10-foldwith PBS, reconcentrated 10-fold by Centricon 30, diluted 10-fold withPBS, and finally reconcentrated 10-fold. The antibody was stored in 0.25ml aliquots at −20° C.

C. Affinity Measurements of he3 IgG for CD5

The affinity of he3 IgG for CD5 was determined using Molt-4M cells,which express CD5 on their surface, and I¹²⁵-labeled chimeric H65 IgG ina competitive binding assay. Culture supernatants from Clone C1705 andC1718 and purified IgG from C1705 were used as the sources of he3 IgG.

For this assay, 20 μg of chimeric H65 IgG (cH65 IgG) was iodinated byexposure to 100 μl lactoperoxidase-glucose oxidase immobilized beads(Enzymobeads, BioRad), 100 μl of PBS, 1.0 mCi I¹²⁵ (Amersham, IMS30), 50μl of 55 mM b-D-glucose for 45 minutes at 23° C. The reaction wasquenched by the addition of 20 μl of 105 mM sodium metabisulfite and 120mM potassium iodine followed by centrifugation for 1 minute to pelletthe beads. ¹²⁵I-cH65 IgG was purified by gel filtration using 7 mls ofsephadex G25, using PBS (137 mM NaCl, 1.47 mM KH₂PO₄, 8.1 mM Na₂HPO₄,2.68 mM KCl at pH 7.2-7.4) plus 0.1% BSA. ¹²⁵I-cH65 IgG recovery andspecific activity were determined by TCA precipitation.

Competitive binding was performed as follows: 100 μl of Molt-4M cellswere washed two times in ice-cold DHB binding buffer (Dubellco'smodified Eagle's medium (Gibco, 320-1965PJ), 1.0% BSA and 10 mM Hepes atpH 7.2.-7.4). Cells were resuspended in the same buffer, plated into 96v-bottomed wells (Costar) at 3×10⁵ cells per well and pelleted at 4° C.by centrifugation for 5 min at 1,000 rpm using a Beckman JS 4.2 rotor;50 μl of 2×-concentrated 0.1 nM ¹²⁵I-cH65 IgG in DHB was then added toeach well and competed with 50 μl of 2×—concentrated cH65 IgG orhumanized antibody in DHB at final antibody concentrations from 100 nMto 0.0017 nM. Humanized antibody was obtained from culture supernatantsof Sp2/0 clone C1718 which expresses he3 IgG. The concentration of theantibody in the supernatants was established by ELISA using a chimericantibody as a standard. The concentration of the antibody in thepurified preparation was determined by binding was allowed to proceed at4° C. for 5 hrs and was terminated by washing cells three times with 200μl of DHB binding buffer by centrifugation for 5 min at 1,000 rpm. Allbuffers and operations were at 4° C. Radioactivity was determined bysolubilizing cells in 100 μl of 1.0 M NaOH and counting in a Cobra IIauto gamma counter (Packard) Data from binding experiments were analyzedby the weighted nonlinear least squares curve fitting program,MacLigand, a Macintosh version of the computer program “Ligand” fromMunson, Analyt. Biochem., 107:220 (1980). Objective statistical criteria(F, test, extra sum squares principle) were used to evaluate goodness offit and for discriminating between models. Nonspecific binding wastreated as a parameter subject to error and was fitted simultaneouslywith other parameters.

Data showing relative binding of he3 and CE65 to CD5 on molt-4M cells ina competition binding assay demonstrate that the moderate-risk changesmade in he3 IgG result in an antibody with a higher affinity than thechimeric mouse-human form of this antibody (cH65) for its target, CD5.

EXAMPLE 7

Preparation of Gelonin Immunoconjugates

Gelonin analogs of the invention were variously conjugated to murine(ATCC HB9286) and chimeric H65 (cH65) antibody, cH65 antibody domains(including cFab, cFab′ and cF(ab′)₂ fragments), and humanized antibodiesand antibody domains, all of which are specifically reactive with thehuman T cell determinant CD5. H65 antibody was prepared and purified bymethods described in U.S. patent application Ser. No. 07/306,433, supraand International Publication No. WO 89/06968, supra. Chimeric H65antibody was prepared according to methods similar to those described inRobinson et al., Human Antibodies and Hybridomas, 2:84-93 (1991),incorporated by reference herein. Chimeric H65 Fab, Fab′, and F(ab′)₂proteins were prepared as described in Better, et al., Proc. Nat. Acad.Sci. (USA), 90: 457-461 (1993), incorporated by reference herein.Finally, he3 humanized antibodies were prepared according to theprocedures described in U.S. patent application Ser. No. 07/808,464,incorporated by reference herein.

A. Conjugation to H65 Antibodies

To expose a reactive sulfhydryl, the unpaired cysteine residues of thegelonin analogs were first reduced by incubation with 0.1 to 2 mM DTT(30-60 minutes at room temperature), and then were desalted bysize-exclusion chromatography.

Specifically, the Gel_(C248) analog (3.8 mg/ml) was treated with 2 mMDTT for 60 minutes in 0.1 M Naphosphate, 0.25 M NaCl, pH 7.5 buffer. TheGel_(C244) variant (7.6 mg/ml) was treated with 2 mM DTT for 30 minutesin 0.1 M Naphosphate, 0.25 M NaCl, pH 7.5 buffer. The Gel_(C247) analog(4 mg/ml) was treated with 2 mM DTT for 30 minutes in 0.1 M Naphosphate,0.5 M NaCl, pH 7.5 buffer with 0.5 mM EDTA. The Gel_(C239) variant (3.2mg/ml) was treated with 2 mM DTT for 30 minutes in 0.1 m Naphosphate,0.5 M NaCl, pH 7.5 buffer with 0.5 mM EDTA. The Gel_(A50(C44)) analog(4.2 mg/ml) was treated with 0.1 mM DTT for 30 minutes in 0.1 MNaphosphate, 0.1 M NaCl, pH 7.5 buffer with 0.5 mM EDTA. Lastly, theGel_(C10) variant (3.1 mg/ml) was treated with 1 mM DTT for 20 minutesin 0.1 M Naphosphate, 0.1 M NaCl, pH 7.5 buffer with 1 mM EDTA.

The presence of a free sulfhydryl was verified by reaction with DTNB andthe average value obtained was 1.4±0.65 SH/molecule. No free thiols weredetected in the absence of reduction.

H65 antibody and chimeric H65 antibody were chemically modified with thehindered linker 5-methyl-2-iminothiolane (M2IT) at lysine residues tointroduce a reactive sulfhydryl group as described in Goff et al.,Bioconjugate Chem., 1:381-386 (1990) and co-owned Carroll et al., U.S.Pat. No. 5,093,475, incorporated by reference herein.

Specifically, for conjugation with Gel_(C248) and Gel_(C244), murine H65antibody at 4 mg/mL was derivitized with 18× M2IT and 2.5 mM DTNB in 25mM TEOA, 150 mM NaCl, pH 8 buffer for 1 hour at 23° C. The reaction gave1.9 linkers per antibody as determined by DTNB assay.

For conjugation with Gel_(C247) and Gel_(C239), H65 antibody at 4.7mg/mL was derivitized with 20× M2IT and 2.5 mM DTNB in 25 mM TEOA 150 mMNaCl, pH 8 buffer for 50 minutes at 23° C. The reaction gave 1.6 linkersper antibody as determined by DTNB assay.

Before reaction with Gel_(A50(C44)), H65 antibody at 5.8 mg/mL wasderivitized with 20× m2IT and 2.5 mM DTNB in 25 mM TEOA, 150 mM NaCl, pH8 buffer for 30 minutes at 23° C. The reaction gave 1.5 linkers perantibody as determined by DTNB assay.

For conjugation with Ge_(C10), H65 antibody at 2.2 mg/mL was derivitizedwith 10× m2IT and 2.5 mM DTNB in 25 mM TEOA, 150 mM NaCl, pH 8 bufferfor 1 hour at 23° C. The reaction gave 1.4 linkers per antibody asdetermined by DTNB assay.

Chimeric H65 antibody was prepared for conjugation in a similar mannerto murine H65 antibody.

Two methods were initially compared for their effectiveness in preparingimmunoconjugates with recombinant gelonin. First, the native disulfidebond in recombinant gelonin was reduced by the addition of 2 mM DTT atroom temperature for 30 minutes. The reduced gelonin was recovered bysize-exclusion chromatography on a column of Sephadex GF-05LS andassayed for the presence of free sulfhydryls by the DTNB assay. 1.4 freeSH groups were detected. This reduced gelonin was then reacted withH65-(M2IT)-S-S-TNB (1.8 TNB groups/H65). Under these experimentalconditions, little or no conjugate was prepared between reduced geloninand thiol-activated H65 antibody.

In contrast, when both the recombinant gelonin and the H65 antibody werefirst derivitized with the crosslinker M2IT (creating gelonin-(M2IT)-SHand H65-(M2IT)-S-S-TNB) and then mixed together,H65-(M2IT)-S-S-(M2IT)-gelonin conjugate was prepared in good yield(toxin/antibody ratio of 1.6). The starting materials for thisconjugation (gelonin-(M2IT)-SH and H65-(M2IT)-S-S-TNB) containedlinker/protein ratios of 1.2 and 1.4, respectively. Native gelonin wasderivatized in a similar manner prior to conjugation to murine orchimeric H65 antibody.

The reduced gelonin analogs were mixed with H65-(M2IT)-S-S-TNB to allowconjugation. The following conjugation reactions were set up for eachanalog: 23 mg (in 7.2 ml) of H65-M2IT-TNB were mixed with a 5-fold molarexcess of Gel_(C248) (23 mg in 6 ml) for 2 hours at room temperature,then for 18 hours overnight at 4° C.; 23 mg (in 7.3 ml) of H65-m2IT-TNBwere mixed with a 5-fold molar excess of Gel_(C244) (23 mg in 3 ml) for3 hours at room temperature, then for 18 hours overnight at 4° C.; 9 mg(in 2.8 mL) of H65-m2IT-TNB were mixed with a 5-fold molar excess ofGel_(C247) (9 mg in 2.25 mL) for 2 hours at room temperature, then for 5nights at 4° C.; 9 mg (in 2.8 mL) of H65-m2IT-TNB were mixed with a5-fold molar excess of Gel_(C239) (9 mg in 2.6 mL) for 2 hours at roomtemperature, then at 4° C. for 3 days; 12 mg (in 1.9 mL) of H65-m2IT-TNBwere mixed with a 5.6-fold molar excess of Gel_(A50(C44)) (13.44 mg in3.2 mL) for 4.5 hours at room temperature, then 4° C. overnight; and 11mg of H65-m2IT-TNB were mixed with a 5-fold molar excess of Gel_(C10)(11 mg in 3.5 mL) for 4 hours at room temperature, then at 4° C.overnight.

Following conjugation, unreacted M2IT linkers on the antibody werequenched with 1:1 mole cysteamine to linker for 15 minutes at roomtemperature. The quenched reaction solution was then loaded onto a gelfiltration column [Sephadex G-150 (Pharmacia) in the case of Gel_(C248),Gel_(C247), Gel_(C244) and Gel_(C239) and an AcA-44 column (IBFBiotecnics, France) in the case of Gel_(A50(C44)) and Gel_(C10)]. Thereactions were run over the gel filtration columns and eluted with 10 mMTris, 0.15M NaCl pH 7. The first peak off each column was loaded ontoBlue Toyopearl® resin (ToSoHaas, Philadelphia, Pa.) in 10 mM Tris, 30 mMNaCl, pH 7 and the product was eluted with 10 mM Tris, 0.5 M NaCl, pH7.5.

Samples of the final conjugation products were run on 5% non-reduced SDSPAGE, Coomassie stained and scanned with a Shimadzu laser densitometerto quantitate the number of toxins per antibody (T/A ratio). The yieldof final product for each analog conjugate was as follows: Gel_(C248),17 mg with a T/A ration of 1.6; Gel_(C247), 1.1 mg with a T/A ratio of1; Gel_(C244), 4.5 mgs with a T/A ratio of 1.46; Gel_(C239), 2.9 mg witha T/A ratio of 2.4; Gel_(A50(C44)), 7.3 mg with a T/A ratio of 1.22; andGel_(C10), 6.2 mg with a T/A ratio of 1.37. Conjugation efficiency(i.e., conversion of free antibody to immunoconjugate) was significantlygreater (˜80%) for some analogs (Gel_(C10), Gel_(A50(C44)), Gel_(C239),Gel_(C247), and Gel_(C248)) than for others (˜10%, Gel_(C244)).

B. Gelonin Immunoconjugates with Chimeric and Humanized Antibodies

Analogs Gel_(C247), and Gel_(A50(C44)), were also conjugated to variouschimeric [cH65Fab, cH65Fab′ and cH65F(ab′)₂], and “human engineered”[he1 Fab, he2-Fab, he3-Fab, he1 Fab′ and he1 F(ab′)₂], antibodyfragments. Chimeric H65 antibody fragments may be prepared according tothe methods described in International Publication No. WO 89/00999,supra. The DNA sequences encoding the variable regions of H65 antibodyfragments that were human engineered (referring to the replacement ofselected murine-encoded amino acids to make the H65 antibody sequencesless immunogenic to humans) according to the methods described above inExample 5, are set out in SEQ ID NO: 69 (variable region of the kappachain of he1 and he2), SEQ ID NO: 70 (variable region of the gamma chainof he1), SEQ ID NO: 71 (variable region of the gamma chain of he2 andhe3) and SEQ ID NO: 72 (variable region of the kappa chain of he3).

The chimeric H65 antibody fragments were conjugated to the Gel_(C247)analog in the same manner as described below for conjugation of humanengineered Fab and Fab′ fragments to Gel_(C247) and Gel_(A50(C44)).

(i) he1 Fab-Gel_(C247)

The he1 Fab was dialyzed into 25 mM TEOA buffer, 250 mM NaCl, pH 8 andthen concentrated to 6.8 mg/mL prior to derivitization with the M2ITcrosslinker. For the linker reaction, M2IT was used at 20-fold molarexcess, in the presence of 2.5 mM DTNB. The reaction was allowed toproceed for 30 minutes at room temperature, then desalted on GF05 (gelfiltration resin) and equilibrated in 0.1 M Na phosphate, 0.2M NaCl, pH7.5. A linker number of 1.8 linkers per Fab was calculated based on theDTNB assay. The he1 Fab-M2IT-TNB was concentrated to 3.7 mg/mL prior toconjugation with Gel_(C247).

Gel_(C247) at 12.8 mg/mL in 10 mM Na phosphate, 0.3M NaCl, was treatedwith 1 mM DTT, 0.5 mM EDTA for 20 minutes at room temperature to exposea reactive sulfhydryl for conjugation and then was desalted on GF05 andequilibrated in 0.1 M Na phosphate, 0.2 M NaCl, pH 7.5. Free thiolcontent was determined to be 0.74 moles of free SH per mole ofGel_(C247) using the DTNB assay. The gelonin was concentrated to 8.3mg/mL prior to conjugation with activated antibody.

The conjugation reaction between the free thiol on Gel_(C247) and thederivitized he1 Fab-M2IT-TNB, conditions were as follows. A 5-foldexcess of the gelonin analog was added to activated he1 Fab-M2IT-TNB(both proteins were in 0.1M Na phosphate, 0.2M NaCl, pH7.5) and thereaction mixture was incubated for 3.5 hours at room temperature andthen overnight at 4° C. Following conjugation, untreated M2IT linkerswere quenched with 1:1 mole cysteamine to linker for 15 minutes at roomtemperature. The quenched reaction solution was loaded onto a gelfiltration column (G-75) equilibrated with 10 mM Tris, 150 mM NaCl, pH7. The first peak off this column was diluted to 30 mM NaCl with 10 mMTris, pH7 and loaded on Blue Toyopearl®. The product was eluted with 10mM Tris, 0.5 M NaCl, pH 7.5.

(ii) he1 Fab′-Gel_(C247)

Similarly, the H65 he1 Fab′ fragment was dialyzed into 25 mM TEOAbuffer, 400 mM NaCl, pH 8 at 2.9 mg/mL prior to derivitization with theM2IT crosslinker. For the linker reaction, M2IT was used at 20-foldmolar excess, in the presence of 2.5 mM DTNB. The reaction was allowedto proceed for 1 hour at room temperature then it was desalted on GF05(gel filtration resin) and equilibrated in 0.1 M Na phosphate, 0.2 MNaCl, pH 7.5. A linker number of 1.6 linkers per Fab′ was calculatedbased on the DTNB assay. The he1 Fab′-M2IT-TNB was concentrated to 3.7mg/mL prior to conjugation with Gel_(C247).

The Ge_(C247) at 77 mg/mL was diluted with 10 mM Na phosphate, 0.1 MNaCl to a concentration of 5 mg/mL, treated with 1 mM DTT, 0.5 mM EDTAfor 30 minutes at room temperature to expose a free thiol forconjugation and then was desalted on GF05 and equilibrated in 0.1 M Naphosphate, 0.2 M NaCl, pH 7.5. Free thiol content was determined to be1.48 moles of free SH per mole of Gel_(C247) using the DTNB assay. TheGel_(C247) was concentrated to 10 mg/mL prior to conjugation withactivated he1 Fab′-M2IT-TNB.

For the reaction between the free thiol on Gel_(C247) and thederivitized he1 Fab′-M2IT-TNB, conditions were as follows. A 5.7-foldmolar excess of gelonin was added to activated hel Fab′-M2IT-TNB and thefinal salt concentration was adjusted to 0.25 M. The reaction mix wasincubated for 1.5 hours at room temperature and then over the weekend at4° C. Following conjugation, unreacted M2IT linkers were quenched with1:1 mole cysteamine to linker for 15 minutes at room temperature. Thequenched reaction solution was loaded onto a gel filtration column(AcA54) equilibrated with 10 mM Tris, 250 mM NaCl, pH 7.5. The firstpeak off this column was diluted to 20 mM NaCl with 10 mM Tris, pH 7 andloaded on Blue Toyopearl® which was equilibrated in 10 mM Tris, 20 mMNaCl, pH 7. The column was then washed with 10 mM Tris, 30 mM NaCl, pH7.5. The product was eluted with 10 mM Tris, 1 M NaCl, pH 7.5.

(iii) he2-Fab Gel_(A50(C44))

The he2-Fab was dialyzed overnight into 25 mM TEOA, 0.25 M NaCl, pH 8buffer and then concentrated to 13.3 mg/mL prior to derivitization withthe M2IT crosslinker. For the linker reaction, M2IT was used in a20-fold molar excess in the presence of 2.5 mM DTNB. The reaction wasallowed to proceed for 20 minutes at room temperature and was thendesalted on a GF05-LS (gel filtration) column, equilibrated in 0.1 M Naphosphate, 0.2 M NaCl with 0.02% Na azide. A linker number of 1.7linkers per Fab-M2IT-TNB was calculated based on the DTNB assay. Afterderivitization and gel filtration, the he2-Fab concentration was 5.2mg/mL.

Gel_(A50(C44)) at 8.33 mg/mL in 10 mM Na phosphate, pH 7.2 was treatedwith 5 mM DTT and 0.5 mM EDTA for 30 minutes at room temperature toexpose a reactive thiol for conjugation and then was desalted on GF05-LSresin equilibrated in 0.1 M Na phosphate, 0.1 M NaCl with 0.5 mM EDTAplus 0.02% Na azide, pH 7.5. Free thiol content was determined to be0.83 moles of free SH per mole of Gel_(A50(C44)) using the DTNB assay.The gelonin was concentrated to 11.4 mg/mL prior to conjugation withactivated he2-Fab.

The conjugation reaction conditions between the free thiol onGel_(A50(C44)) and the derivitized he2-Fab-M2IT-TNB were as follows. A3-fold excess of the gelonin analog was added to activatedhe2-Fab-M2IT-TNB (both proteins were in 0.1 M Na phosphate, 0.1 M NaCl,pH 7.5 but the gelonin solution contained 0.5 mM EDTA as well). Thereaction mixture was concentrated to half its original volume, then themixture was incubated for 4 hours at room temperature followed by 72hours at 4° C. Following the incubation period the efficiency ofconjugation was estimated at 70-75% by examination of SDS PAGE.

Following conjugation the excess M2IT linkers were quenched byincubation with 1:1 mole cysteamine to linker for 15 minutes at roomtemperature. The quenched reaction as loaded onto a gel filtrationcolumn (G-75) equilibrated in 10 mM Tris, 0.15 M NaCl, pH 7. The firstpeak off this column was diluted to 30 mM NaCl with 10 mM Tris, pH 7 andloaded onto a Blue Toyopearl® (TosoHaas) column. The product was elutedwith 10 mM Tris, 1 M NaCl, pH 7.5.

(iv) he3-Fab Gel_(A50(C44))

Similarly, the he3-Fab was dialyzed overnight into 25 mM TEOA, 0.25 MNaCl, pH 8 buffer and then concentrated to 5 mg/mL prior toderivitization with the M2IT crosslinker. For the linker reaction, M2ITwas used in a 10-fold molar excess in the presence of 2.5 mM DTNB. Thereaction was allowed to proceed for 45 minutes at room temperature andwas then desalted on a GF05-LS (gel filtration) column, equilibrated in0.1 M Na phosphate, 0.2 M NaCl with 0.02% Na azide. A linker number of 1M2IT per Fab-M2IT-TNB was calculated based on the DTNB assay. Afterderivitization and gel filtration, the he3-Fab concentration was 5.3mg/mL.

Gel_(A50(C44)) at 7.8 mg/mL in 0.1 M Na phosphate, 0.1 M NaCl, pH 7.5was treated with 1.5 mM DTT and 1 mM EDTA for 30 minutes at roomtemperature to expose a reactive thiol for conjugation and then wasdesalted on GF05-LS resin equilibrated in 0.1 M Na phosphate, 0.1 M NaClplus 0.02% Na azide, pH 7.5. Free thiol content was determined to be0.66 moles of free SH per mole of Gel_(A50(C44)) using the DTNB assay.The gelonin was concentrated to 5.2 mg/mL prior to conjugation withactivated he3-Fab.

The conjugation reaction conditions between the free thiol onGel_(A50(C44)) and the derivitized he3-Fab-M2IT-TN were as follows. A5-fold excess of the gelonin analog was added to activatedhe3-Fab-M2IT-TNB (both proteins were in 0.1 M Na phosphate 0.1 M NaCl,pH 7.5). The reaction mixture was incubated for 2 hours at roomtemperature followed by 72 hours at 4° C. Following the incubated periodthe efficiency of conjugation was estimated at 70-75% by examination ofSDS PAGE.

Following conjugation, the excess M2IT linkers were quenched byincubation with 1:1 mole cysteamine to linker for 15 minutes at roomtemperature. The quenched reaction was loaded onto a GammaBind G(immobilized protein G affinity resin, obtained from Genex,Gaithersburg, Md.) equilibrated in 10 mM Na phosphate, 0.15 M NaCl, pH7. It was eluted with 0.5 M NaOAc, pH 3 and neutralized with Tris. Itwas dialyzed into 10 mM Tris, 0.15 M NaCl, pH 7 overnight, then dilutedto 30 mM NaCl with 10 mM Tris, pH 7 and loaded onto a blue Toyopearl®(TosoHaas) column. The product was eluted with 10 mM Tris, 1 M NaCl, pH7.5.

EXAMPLE 8

Whole Cell Kill Assays

Immunoconjugates prepared with gelonin and gelonin analogs were testedfor cytotoxicity against an acute lymphoblastoid leukemia T cell line(HSB2 cells) and against human peripheral blood mononuclear cells(PBMCs). Immunoconjugates of ricin A-chain with H65 antibody (H65-RTA)and antibody fragments were also tested. The ricin A-chain (RTA) as wellas the H65-RTA immunoconjugates were prepared and purified according tomethods described in U.S. patent application Ser. No. 07/306,433, supraand in International Publication No. WO 89/06968, supra.

Briefly, HSB2 cells were incubated with immunotoxin and the inhibitionof protein synthesis in the presence of immunotoxin was measuredrelative to untreated control cells. The standard immunoconjugatesH65-RTA (H65 derivitized with SPDP linked to RTA), H65-Gelonin andH65-rGelonin, H65 fragment immunoconjugate, and gelonin immunoconjugatesamples were diluted with RPMI without leucine at half-logconcentrations ranging from 2000 to 0.632 ng/ml. All dilutions wereadded in triplicate to wells of microtiter plates containing 1×10⁵ HSB2cells per well. HSB2 plates were incubated for 20 hours at 37° C. andthen pulsed with ³H-Leu for 4 hours before harvesting. Samples werecounted on the Inotec Trace 96 cascade ionization counter. By comparisonwith an untreated sample, the picomolar concentration (pM) ofimmunotoxin which resulted in a 50% inhibition of protein synthesis(IC₅₀) was calculated. In order to normalize for conjugates containingdiffering amounts of toxin or toxin analog, the cytotoxicity data wereconverted to picomolar toxin (pM T) by multiplying the conjugate IC₅₀(in pM) by the toxin/antibody ratio which is unique to each conjugatepreparation.

The PMBC assays were performed as described by Fishwild et al., Clin.and Exp. Immunol., 86:506-513 (1991) and involved the incubation ofimmunoconjugates with PBMCs for a total of 90 hours. During the final 16hours of incubation, ³H-thymidine was added; upon completion,immunoconjugate-induced inhibition of DNA synthesis was quantified. Theactivities of the H65 and chimeric H65 antibody conjugates against HSB2cells and PBMC cells are listed in Table 2 below.

TABLE 2 IC₅₀ (pM T) Conjugate HSB2 Cells PBMCs H65-RTA 143 459H65-(M2IT)-S-S-(M2IT)-Gelonin 1770 81 H65-(M2IT)-S-S-(M2IT)-rGelonin 27675 H65-(M2IT)-S-S-Gel_(C10) 140 28 H65-(M2IT)-S-S-Gel_(A50(C44)) 99 51H65-(M2IT)-S-S-Gel_(C239) 2328 180 H65-(M2IT)-S-S-Gel_(C244) >5000 >2700H65-(M2IT)-S-S-Gel_(C247) 41 35 H65-(M2IT)-S-S-Gel_(C248) 440 203cH65-RTA₃₀ 60 400 cH65-(M2IT)-S-S-(M2IT)-Gelonin 1770 140cH65-(M2IT)-S-S-(M2IT)-rGelonin 153 120 cH65-(M2IT)-S-S-Gel_(C239) >7000290 cH65-(M2IT)-S-S-Gel_(C247) 34 60 cH65-(M2IT)-S-S-Gel_(C248) 238 860H65-(M2IT)-S-S-Gel_(A44(C50)) 338 ND* H65-(M2IT)-S-S-Gel_(C247A44A50) 71ND* *Not determined.

Against HSB2 cells, many of the gelonin analog immunoconjugates weresignificantly more potent than conjugates prepared with native geloninor recombinant, unmodified gelonin, both in terms of a low IC₅₀ value,but also in terms of a greater extent of cell kill. Against human PBMCs,the gelonin analog conjugates were at least as active as native andrecombinant gelonin conjugates. Importantly, however, some of theconjugates (for example, Gel_(C10), Gel_(A50(C44)) and Gel_(C247))exhibited an enhanced potency against PBMCs compared to native andrecombinant gelonin conjugates, and also exhibited an enhanced level ofcell kill.

The activities of the H65 antibody fragment conjugates against HSB2cells and PBMC cells are listed in Tables 3 and 4 below, wherein extentof kill in Table 3 refers to the percentage of protein synthesisinhibited in HSB2 cells at the highest immunotoxin concentration tested(1 μg/ml).

TABLE 3 IC₅₀ (pM T) Conjugate HSB2 Cells PBMCs cH65Fab′-RTA 30 530 1800cH65Fab′-rGelonin 135 160 cH65Fab′-Gel_(C247) 48 64 cH65F(ab′)₂-RTA 3033 57 cH65F(ab′)₂-rGelonin 55 34 cH65F(ab′)₂-Gel_(C247) 23 20cH65F(ab′)₂-Gel_(C248) 181 95

TABLE 4 IC₅₀ (pM T) Conjugate HSB2 Cells Extent of Kill he1Fab′-Gel_(C247) 57.7 93% he1 Fab-Gel_(C247) 180.0 94%he2-Fab-Gel_(A50(C44)) 363.0 91% he3-Fab-Gel_(A50(C44)) 191.0 93%cH65Fab′-Gel_(C247) 47.5 93% cH65F(ab′)₂-rGelonin 45.4 85%cH65F(ab′)₂-Gel_(C247) 77.5 83% cH65F(ab′)₂-Gel_(C247) 23.2 85%

The data in Table 3 show that monovalent (Fab or Fab′) fragmentsconjugated to various forms of gelonin are more potent than RTAconjugates. Table 4 shows that the human-engineered gelonin-Fabconjugates exhibit a very high degree of extent of kill.

EXAMPLE 9

Properties of Gelonin Immunoconjugates

A. Solubility

Recombinant gelonin and the gelonin analogs exhibited enhancedsolubility in comparison to both native gelonin and RTA30. In addition,recombinant gelonin and gelonin analog immunoconjugates exhibitedenhanced solubility relative to immunoconjugates prepared with nativegelonin and RTA30. This enhanced solubility was particularly noteworthyfor recombinant gelonin and analog conjugates prepared with chimeric Fabfragments.

B. Disulfide Bond Stability Assay

The stability of the disulfide bond linking a RIP to a targetingmolecule (such as an antibody) is known to influence the lifespan ofimmunoconjugates in vivo [See Thorpe et al., Cancer Res., 47:5924-5931(1987), incorporated by reference herein]. For example, conjugates inwhich the disulfide bond is easily broken by reduction in vitro are lessstable and less efficacious in animal models [See Thorpe at al., CancerRes., 48:6396-6403 (1988), incorporated by reference herein].

Immunoconjugates prepared with native gelonin, recombinant gelonin andgelonin analogs were therefore examined in an in vitro disulfide bondstability assay similar to that described in Wawrzynczak et al., CancerRes., 50:7519-7526 (1990), incorporated by reference herein. Conjugateswere incubated with increasing concentrations of glutathione for 1 hourat 37° C. and, after terminating the reaction with iodoacetamide, theamount of RIP released was quantitated by size-exclusion HPLC on aTosoHaas TSK-G2000SW column.

By comparison with the amount of RIP released by high concentrations of2-mercaptoethanol (to determine 100% release), the concentration ofglutathione required to release 50% of the RIP (the RC₅₀) wascalculated. The results of assays for H65 antibody conjugates are setout in Table 5 below.

TABLE 5 Conjugate RC₅₀ (mM) H65-RTA 30 3.2 H65-(M2IT)-S-S-(M2IT)-gelonin11.1 H65-(M2IT)-S-S-(M2IT)-rGelonin 3.0 H65-(M2IT)-S-S-Gel_(C10) 2.5H65-(M2IT)-S-S-Gel_(A50(C44)) 0.6 H65-(M2IT)-S-S-Gel_(C239) 774.0H65-(M2IT)-S-S-Gel_(C244) 1.2 H65-(M2IT)-S-S-Gel_(C247) 0.1H65-(M2IT)-S-S-Gel_(C248) 0.4 cH65-RTA 30 2.50cH65-(M21T)-S-S-(M2IT)-rGelonin 2.39 cH65-(M21T)-S-S-Gel_(C247) 0.11cH65-(M2IT)-S-S-Gel_(C248) 0.32 H65-(M2IT)-S-S-Gel_(A44(C50)) 9.2H65-(M2IT)-S-S-Gel_(C247A44A50) 0.3

The foregoing results indicate that the stability of the bonds betweenthe different gelonin proteins and H65 antibody varied greatly. With theexception of Gel_(C10) and Gel_(C239), most of the gelonin analogsresulted in conjugates with linkages that were somewhat less stable inthe in vitro assay than the dual-linker chemical conjugate. Thestability of the Gel_(C239) analog, however, was particularly enhanced.

The results of the assay for H65 antibody fragment conjugates are setout in Table 6 below.

TABLE 6 Conjugate RC₅₀ (mM) he1 Fab′-Gel_(C247) 0.07 cFab′-Gelonin 1.27cFab′-Gel_(C247) 0.08 cF(ab′)₂-RTA 30 1.74 cF(ab′)₂-rGelonin 2.30cF(ab′)₂-Gel_(C247) 0.09 cF(ab′)₂-Gel_(C248) 0.32 he2-Fab-Gel_(A50(C44))0.46 he3-Fab-Gel_(A50(C44)) 0.58

From the RC₅₀ results presented in Tables 5 and 6, it appears that theparticular RIP analog component of each immunotoxin dictates thestability of the immunotoxin disulfide bond in vitro.

EXAMPLE 10

Pharmacokinetics of Conjugates to H65 Antibody

The pharmacokinetics of gelonin analogs Gel_(C247), Gel_(A50(C44)), andGel_(C10) linked to whole H65 antibody was investigated in rats. An IVbolus of 0.1 mg/kg of ¹²⁵I-labelled immunoconjugateH65-(M2IT)-S-S-Gel_(C247), H65-(M2IT)-S-S-Gel_(A50(C44)) orH65-(M2IT)-S-S-Gel_(C10) was administered to male Sprague-Dawley ratsweighing 134-148 grams. Serum samples were collected from the rats at 3,15, 30 and 45 minutes, and at 1.5, 2, 4, 6, 8, 18, 24, 48, 72, and 96hours. Radioactivity (cpm/ml) of each sample was measured, and SDS-PAGEwas performed to determine the fraction of radioactivity associated withwhole immunoconjugate. Immunoconjugate-associated serum radioactivitywas analyzed using the computer program PCNONLIN (SCI Software,Lexington, Ky.). Table 7 below lists the pharmacokinetic parameters ofthe immunoconjugates. In that table, the standard error for each valueis indicated and a one way analysis of variance is presented, IC is theimmunoconjugate (specified by the abbreviation for the gelonin variantthat is part of the immunoconjugate), n is the number of animals in thestudy, Vc is the central volume of distribution, Cl is the clearance,MRT is the total body mean residence time, Alpha is the α half-life andBeta is the β half-life of the immunoconjugate.

TABLE 7 Vc Cl MRT Alpha Beta IC (ml/kg) (ml/hr/kg) (hours) (hours)(hours) H65 Gel_(C247) 65.3 ± 11.0 ± 0.4 16.5 ± 2.3 ± 0.2 20.5 ± 3.0 n =32 3.4 1.9 H65 Gel_(A50(C44)) 61.9 ± 4.1 ± 0.1 22.7 ± 3.0 ± 0.7 17.8 ±0.8 n = 38 2.4 0.7 H65 Gel_(C10) 59.2 ± 2.5 ± 0.04 42.7 ± 3.3 ± 0.3 32.9± 1.1 n = 45 1.3 1.1 p-value 0.176 <0.0001 <0.0001 0.303 <0.0001

The Gel_(C247) immunoconjugate was found to have α and β half lives of2.3 and 20 hours, with a total mean residence time of 17 hours. The 72and 96 hour time points were excluded from analysis because of the poorresolution of immunoconjugate associated radioactivity on the SDS-PAGEgel for these serum samples.

Because in vitro studies suggested that the Gel_(C10) immunoconjugatehad greater disulfide bond stability it was anticipated that its halflives in vivo would be longer relative to the cys₂₄₇ form of theimmunoconjugate. The β half life of the immunoconjugate was about 33hours compared to 20 hours for the Gel_(C247) conjugate. The total meanresidence time was also much greater for the Gel_(C10) immunoconjugate(42 hours versus 42 hours for the Gel₂₄₇ conjugate). In addition, theclearance of the Gel_(C10) immunoconjugate was 2.5 ml/hr/kg, about fourtimes less than that of the Gel_(C247) immunoconjugate (11 ml/hr/kg). Asalso predicted from the in vitro disulfide stability data, the clearanceof the Gel_(A50(C44)) immunoconjugate was intermediate between those ofthe Gel_(C10) and Gel_(C247) immunoconjugates.

Based on these studies, the Gel_(C10) analog conjugated to H65 antibodyhas greater in vivo stability than the Gel_(A50(C44)) and Gel_(C247)analogs conjugated to H65 antibody (as determined by the longer meanresidence time and clearance rates), although the properties of theGel_(A50(C44)) immunoconjugate more closely resembled those of theGel_(C10) immunoconjugate than the Gel_(C247) immunoconjugate.

EXAMPLE 11

Pharmacokinetics of Conjugates to H65 Antibody Fragments

The pharmacokinetics of Gel_(C247) and Gel_(A50(C44)) analogs linked tohuman engineered H65 Fab fragments were also investigated in rats. An IVbolus of 0.1 mg/kg of ¹²⁵l-labelled he1 H65 Fab-Gel_(C247), he2 H65Fab-Gel_(A50(C44)) or he3 H65 Fab-Gel_(A50(C44)) was administered tomale Sprague-Dawley rats weighing 150-180 grams. Serum samples werecollected at 3, 5, 15, 20, 30, and 40 minutes, and 1, 1.5, 3, 6, 8, 18,24, 32, 48, and 72 hours, and were analyzed by ELISA using rabbitanti-Gelonin antibody as the capture antibody and biotin-labelled goatanti-human kappa light chain antibody as the secondary antibody. Resultsof the analysis are presented in Table 8 below. In the table, thestandard error for each value is shown, and IC is the immunoconjugate, nis the number of animals in the study, Vc is the central volume ofdistribution, Vss is the steady state volume of distribution, Cl is theclearance, MRT is the total body mean residence time, Alpha is the αhalf-life and Beta is the β half-life of the indicated conjugate.

TABLE 8 Vss Cl VC (ml/ (ml/ MRT Alpha Beta IC (ml/kg) hr/kg) hr/kg)(hours) (hours) (hours) hel Gel_(C247) 48 ± 3 133 ± 7 62 ± 3 2.1 ± 0.33± 3.0 n = 27 0.1 0.03 fixed he2 54 ± 5 141 ± 8 53 ± 3 2.7 ± 0.37 ± 3.1Gel_(A50(C44)) 0.2 0.04 fixed n = 28 he3 77 ± 6 140 ± 57 ± 3 2.5 ± 0.58± 3.0 ± Gel_(A50(C44))  20 0.4 0.11 1.0 n = 33

Comparing the three immunoconjugates, the pharmacokinetics of he1 H65Fab-Gel_(C247), he2 H65 Fab-Gel_(A50(C44)) and he3-Fab-Gel_(A50(C44))were very similar, having similar alpha and beta half-lives, meanresidence times, and clearance, particularly when comparing parametersobtained from the ELISA assayed curves. This is in contrast to theirwhole antibody immunoconjugate counterparts, where the clearance ofGel_(C247) immunoconjugate (11 ml/kg/hr) was three-fold greater thanthat of Gel_(A50(C44)) immunoconjugate (4 ml/kg/hr). This suggests thatcleavage of the disulfide bond linking the Fab fragment and gelonin isnot as important for the serum clearance of Fab immunoconjugates as forwhole antibody immunoconjugates.

EXAMPLE 12

Immunogenicity of Immunoconjugates

Outbred Swiss/Webster mice were injected repeatedly (0.2 mg/kg eachinjection) with murine H65 antibody conjugates prepared with RTA, RTA30and recombinant gelonin. The cycle was such that each animal wasinjected on days 1 and 2, and then the injections were repeated 28 and29 days later. The animals received 5 such cycles of injections. Oneweek and three weeks following each series of injections, blood wascollected and the amount of anti-RIP antibodies present was determinedby ELISA; peak titers for each cycle are shown in Table 9. RTA and RTA30generated strong responses which began immediately following the firstcycle of injections and remained high throughout the experiment. Incontrast, no immune response was detected for the gelonin conjugate,even after 5 cycles of injections. When the conjugates were mixed withComplete Freund Adjuvant and injected i.p. into mice, anti-RTA andRTA-30 antibodies were readily detected after several weeks. These dataindicate that anti-gelonin antibodies, if generated, would have beendetected by the ELISA assay, and suggest that recombinant gelonin may bemuch less immunogenic in animals than is RTA.

TABLE 9 Cycle H65-RTA H65-RTA30 H65-rGel Prebleed 100 100 100 Cycle 1168 117 100 Cycle 2 4208 1008 100 Cycle 3 7468 3586 100 Cycle 4 57073936 100 Cycle 5 4042 2505 100

EXAMPLE 13

In vivo Efficacy of Immunoconjugates

A human peripheral blood lymphocyte (PBL)-reconstituted, severe combinedimmunodeficient mouse model was utilized to evaluate the in vivoefficacy of various Immunoconjugates comprising the gelonin analogsGel_(C247) and Gel_(A50(C44)). Immunoconjugates were tested for thecapacity to deplete human blood cells expressing the CD5 antigen.

A. Human PBL Donors and Cell Isolation

Human peripheral blood cells were obtained from lymphapheresis samples(HemaCare Corporation, Sherman Oaks, Calif.) or venous blood samples(Stanford University Blood Bank, Palo Alto, Calif.) collected fromhealthy donors. Blood cells were enriched for PBLs using Ficoll-Hypaquedensity gradient centrifugation (Ficoll-Paque®; Pharmacia, Piscataway,N.J.) and subsequently washed 4 times with PBS. Residual erythrocyteswere lysed with RBC lysing buffer (16 μM ammonium chloride, 1 mMpotassium bicarbonate, 12.5 μM EDTA) during the second wash. Cellviability in the final suspension was >95% as assessed by trypan bluedye exclusion.

B. Animals and Human PBL Transfer

CB.17 scid/scid (SCID) mice were purchased from Taconic (Germantown,N.Y.) or were bred under sterile conditions in a specific pathogen-freeanimal facility (original breeding pairs were obtained from HanaBiologics, Alameda, Calif.). Animals were housed in filter-top cages andwere not administered prophylactic antibiotic treatment. Cages, bedding,food and water were autoclaved before use. All manipulations withanimals were performed in a laminar flow hood.

Untreated SCID mice were bled for determination of mouse Ig levels.Human PBL-injected mice were bled at various intervals for quantitationof human Ig and sIL-2R. Blood collection was from the retro-orbitalsinus into heparinized tubes. Blood samples were centrifuged at 300×gfor 10 min, and plasma was collected and stored at −70° C. Mouse andhuman Ig were quantified using standard sandwich ELISAs. Briefly,flat-bottom microtiter plates (MaxiSorp Immuno-Plates, Nunc, Roskilde,Denmark) were coated overnight at 4° C. with goat anti-mouse IgG+IgA+IgM(Zymed Laboratories, Inc., South San Francisco, Calif.) or goatanti-human Igs (Tago, Inc., Burlingame, Calif.) in bicarbonate buffer,pH 9.6. Plates were blocked for 2 hours at room temperature with 1% BSAin Tris-buffered saline, pH 7.5 (TBS), and then incubated at 37° C. for1 hour with standards or samples serially-diluted in TBS/1% BSA/0.05%Tween 20. Standards used were a monoclonal mouse IgG2a (IND1anti-melanoma; XOMA Corporation, Berkeley, Calif.) and polyclonal humanIg (Sigma Chemical Co., St. Louis, Mo.). Subsequently, plates werewashed with TBS/Tween 20 and incubated at 37° C. for 1 hour withalkaline phosphatase-conjugated goat anti-mouse IgG+IgA+IgM or goatanti-human Igs (Caltag Laboratories, South San Francisco, Calif.).Detection was by measurement of absorbance at 405 nm followingincubation with 1 mg/ml p-nitro-phenylphosphate (Sigma) in 10%diethanolamine buffer, pH 9.8. Plasma from a normal BALB/c mouse wasused as a positive control in the mouse Ig ELISA. Plasma samples fromnaive SCID mice or normal BALB/c mice did not have detectable levels ofhuman Ig. Human sIL-2R was quantified using an ELISA kit (ImmunotechS.A., Marseille, France) as per the manufacturer's instructions.

Five-to-seven week old mice with low plasma levels of mouse Ig (<10μg/ml) were preconditioned with an i.p. injection of cyclophosphamide(Sigma) at 200 mg/kg. Two days later, they were injected i.p. with25-40×10⁶ freshly-isolated human PBL suspended in 0.8 ml PBS.

C. Immunoconjugate Treatment

SCID mice were bled at approximately 2 weeks after human PBLtransplantation. Mice with undetectable (<10 pM) or low plasma levels ofhuman sIL-2R were eliminated from the study. The cut-off for exclusionof mice with detectable, but low, levels of human sIL-2R was empiricallydetermined for each study and was generally 20 pM. The remaining micewere divided into groups and were administered vehicle orimmunoconjugate as an i.v. bolus (0.2 mg/kg) daily for 5 consecutivedays. Animals were sacrificed 1 day after cessation of treatment forquantitation of human T cells in tissues and human sIL-2R in plasma.

D. Collection of Tissues and Analysis of PBL Depletion

Blood was collected from the retro-orbital sinus into heparinized tubes.Mice were then killed by cervical dislocation and spleens were removedaseptically. Single cell suspensions of splenocytes were prepared inHBSS by pressing the spleens between the frosted ends of sterile glassmicroscope slides. Collected cells were washed twice with PBS.Erythrocytes were eliminated from blood and splenocyte suspensions usingRBC lysing buffer. Subsequently, cells were resuspended in PBS forenumeration. Recovered cells were then assayed for Ag expression usingflow cytometry.

Two to five hundred thousand cells in 100 μl of PBS/1% BSA/0.1% sodiumazide were incubated on ice for 30 min. with saturating amounts ofvarious FITC- or phycoerythrin (PE)-conjugated Abs (Becton-Dickinson,Mountain View, Calif.) Abs used for staining included: HLe-1-FITC (IgG1anti-CD45), Leu 2-FITC (IgG1 anti-CD8), Leu 3 PE (IgG1 anti-CD4), andLeu M3-PE (IgG2a anti-CD14). Cells were then washed in cold buffer andfixed in 0.37% formaldehyde in PBS. Samples were analyzed on a FACscan(Becton-Dickinson) using log amplifiers. Regions to quantify positivecells were set based on staining of cells obtained from naive SCID mice.The absolute numbers of human Ag-positive cells recovered from SCIDtissues were determined by multiplying the percent positive cells by thetotal number of cells recovered from each tissue sample. The totalnumber of leukocytes in blood was calculated using a theoretical bloodvolume of 1.4 ml/mouse. The detection limit for accurate quantitation ofhuman cells in SCID mouse tissues was 0.05%. All statistical comparisonbetween treatment groups were made using the Mann-Whitney U test.Treatment groups were determined to be significantly different frombuffer control groups when the p value was <0.05. Results are presentedin Table 10 below, wherein + indicates a significant difference fromcontrols, − indicates an insignificant difference and NT means theconjugate was not tested. CD5 Plus (XOMA Corporation, Berkeley, Calif.),is mouse H65 antibody chemically linked to RTA and is a positivecontrol. OX19 Fab-Gel_(C247) is a negative control immunoconjugate. TheOX19 antibody (European Collection of Animal Cell Cultures #84112012) isa mouse anti-rat CD5 antibody that does not cross react with human CD5.

TABLE 10 Human T Cell Depletion Test Article Spleen Blood CD5 Plus + +cH65 F(ab′)₂ − − cH65 Fab′ − − H65-rGEL + + cH65 F(ab′)₂-rGel + + cH65Fab′-rGel + + cH65 F(ab′)₂-Gel_(c247) + NT cH65 Fab′-Gel_(c247) + +he1H65 Fab′-Gel_(c247) + NT cH65 Fab′-Gel_(A50(C44)) + + OX19Fab-Gel_(c247) − −

All the gelonin immunoconjugates were capable of depleting human cellsin the SCID mouse model.

EXAMPLE 14

Construction of Gelonin Immunofusions with Chimeric Antibodies

Several genetic constructs were assembled which included a naturalsequence gelonin gene fused to an H65 truncated heavy chain gene (Fd orFd′), or an H65 light chain gene (kappa). In this Example, H65 Fd, Fd′,and H65 light chain refer to chimeric constructs. The H65 Fd sequenceconsists of the nucleotides encoding the murine H65 heavy chain variable(V), joining (J) and human IgG₁, constant (C) domain 1 regions,including the cysteine bound to light chain IgG₁ and has the carboxylterminal sequence SCDKTHT (SEQ ID NO: 130). The H65 Fd′ sequence has theH65 Fd sequence with the addition of the residues CPP from the hingeregion of human IgG₁ heavy chain, including a cysteine residue which isbound to the other human IgG₁ heavy chain and its F(ab′)₂ fragment. SeeBetter, et al., Proc. Nat. Acad. Sci. (USA), 90: 457-461 (1993),incorporated by reference herein.

The H65 light chain sequence consists of the nucleotides encoding themurine H65 light chain variable (V), joining (J), and human kappa(C_(k)) regions. The DNA sequences of the V and J regions of the H65 Fdand kappa fragment genes linked to the pelB leader can be obtained fromGenBank (Los Alamos National Laboratories, Los Alamos, N. Mex.) underAccession Nos. M90468 and M90467, respectively. Several of the genefusions included a gelonin gene linked at the 5′ end of an H65 Fabfragment gene while the others included a gelonin gene linked at the 3′end of an H65 Fab fragment gene. A DNA linker encoding a peptide segmentof the E. coli shiga-like toxin (SLT) (SEQ ID NO: 56), which containstwo cysteine residues participating in a disulfide bond and forming aloop that includes a protease sensitive amino acid sequence) or ofrabbit muscle aldolase [(RMA) as in SEQ ID NO: 57, which containsseveral potential cathepsin cleavage sites] was inserted between thegelonin gene and the antibody gene in the constructs. Alternatively, adirect fusion was made between a gelonin gene and an H65 Fab fragmentgene without a peptide linker segment. Table 11 below sets out adescriptive name of each gene fusion and indicates the expressionplasmid containing the gene fusion and the section of the application inwhich each is designated. Each plasmid also includes the Fab fragmentgene (shown in parentheses in Table 11) with which each particular genefusion was co-expressed. The inclusion of a cysteine from a hinge region(Fd′) allows potential formation of either monovalent Fab′ or bivalentF(ab′)₂ forms of the expression product of the gene fusion.

TABLE 11 Section Plasmid Description B(i) pING3754 Gelonin::SLT::Fd′(kappa) B(ii) pING3757 Gelonin::SLT::kappa (Fd′) B(iii) pING3759Gelonin::RMA::Fd′ (kappa) B(iv) pING3758 Gelonin::RMA::kappa (Fd′) A(i)pING4406 Fd::SLT::Gelonin (kappa) A(ii) pING4407 kappa::SLT::Gelonin(Fd) A(iii) pING4408 Fd::RMA::Gelonin (kappa) A(iv) pING4410kappa::RMA::Gelonin (Fd) C(i) pING3334 Gelonin::Fd (kappa)

A. Fusions of Gelonin at the Carboxyl-Terminus of Antibody Genes

(i) Fd::SLT::Gelonin (kappa)

A gelonin gene fusion to the 3′-end of the H65 Fd chain with the 23amino acid SLT linker sequence was assembled in a three piece ligationfrom plasmids pVK1, pING3731 (ATCC 68721) and pING4000. Plasmid pVK1contains the Fd gene linked in-frame to the SLT linker sequence and someH65 Fd′ and kappa gene modules as in pING3217, shown in Better, et al.,Proc. Nat. Acad. Sci. (USA): 457-461 (1993), except that the kappa andFd′ regions are reversed. Plasmid pING3731 contains the gelonin gene,and pING4000 contains the H65 kappa and Fd′ genes each linked to thepelB leader sequence under the control of the araB promoter as adicistronic message.

Plasmid pVK1 was designed to link the 3′-end of a human IgG Fd constantregion in-frame to a protease-sensitive segment of the SLT gene boundedby two cysteine residues which form an intra-chain disulfide bond. TheSLT gene segment (20 amino acids from SLT bounded by cysteine residues,plus three amino acids introduced to facilitate cloning) was assembledfrom two oligonucleotides, SLT Linker 1 and SLT Linker 2.

SLT Linker 1 (SEQ ID NO: 73) 5′ TGTCATCATCATGCATCGCGAGTTGCCAGAATGGCATCTGATGAGTTTCCTTCTATGTGCGCAAGTACTC 3′

SLT Linker 2 (SEQ ID NO: 74) 5′ TCGAGAGTACTTGCGCACATAGAAGGAAACTCATCAGATGCCATTCTGGCAACTCGCGATGCATGATGATGACATGCA 3′

The two oligonucleotides were annealed and ligated into a vector(pING3185) containing PstI and XhoI cohesive ends, destroying the PstIsite and maintaining the XhoI site. Plasmid pING3185 contained anengineered PstI site at the 3′-end of the Fd gene, and contained an XhoIsite downstream of the Fd gene. The product of this ligation, pVK1,contained the H65 Fd gene (fused to the pelB leader) in frame with theSLT linker segment, and contained two restriction sites, FspI and ScaI,at the 3′-end of the SLT linker.

Plasmid pVK1 was digested with SauI and ScaI, and the 217 bp fragmentcontaining a portion of the Fd constant domain and the entire SLT genesegment was purified by electrophoresis on an agarose gel. pING3731 wasdigested with SmaI and XhoI and the 760 bp gelonin gene was similarlypurified. Plasmid pING4000 was digested with SauI and XhoI and thevector segment containing the entire kappa gene and a portion of the Fdgene was also purified. Ligation of these three DNA fragments resultedin pING4406 containing the Fd::SLT::Gelonin (kappa) gene fusion vector.

(ii) kappa::SLT::Gelonin (Fd)

A gelonin gene fusion to the 3′-end of the H65 kappa chain with the 25amino acid SLT linker sequence (20 amino acids from SLT bounded bycysteine residues, plus 5 amino acids introduced to facilitate cloning)was assembled from the DNA segments in pING3731 (ATCC 68721) andpING3713.

Plasmid pING3713 is an Fab expression vector where the H65 Fd and kappagenes are linked in a dicistronic transcription unit containing the SLTlinker segment cloned in-frame at the 3′-end of the kappa gene. Theplasmid was constructed as follows. In a source plasmid containing theH65 Fd and kappa genes, an EagI site was positioned at the 3′-end of thekappa gene by site directed mutagenesis without altering the encodedamino acid sequence. The SLT gene segment from pVK1 was amplified withprimers SLT-EagI-5′ and SalI for in frame linkage to the EagI site atthe 3′-end of the kappa gene.

SLT-Eag-5′ (SEQ ID NO: 75) 5′ TGTTCGGCCGCATGTCATCATCATGCATCG 3′

SalI (SEQ ID NO: 76) 5′ AGTCATGCCCCGCGC 3′

The 140 bp PCR product was digested with EagI and XhoI, and the 75 bpfragment containing the SLT gene segment was cloned adjacent to the Fdand kappa genes in the source plasmid to generate pING3713.

For construction of gene fusion to gelonin, pING3713 was cut with ScaIand XhoI, and the vector fragment containing the Fd gene and kappa::SLTfusion was purified. pING3731 was digested with SmaI and XhoI and theDNA fragment containing the gelonin gene was also purified. The productof the ligation of these two fragments, pING4407, contains the Fd andkappa::SLT::gelonin genes.

(iii) Fd::RMA::Gelonin (kappa)

A gelonin gene fusion to the 3′-end of the H65 Fd chain with the 21amino acid RMA linker sequence (20 amino acids from RMA, plus 1 aminoacid introduced to facilitate cloning) was assembled in a three pieceligation from plasmids pSH4, pING3731 (ATCC 68721) and pING4000.

Plasmid pSH4 contains an Fd gene linked in frame to the RMA linkersequence. The RNA gene segment was linked to the 3′-end of Fd by overlapextension PCR as follows. The 3′-end (constant region) of the Fd genewas amplified by PCR from a source plasmid with the primers KBA-γ2 andRMAG-1. Any Fd constant region may be used because constant regions ofall human IgG₁ antibodies are identical in this region.

KBA-γ2 (SEQ ID NO: 77) 5′ TCCCGGCTGTCCTACAGT 3′

RHAG-1 (SEQ ID NO: 78) 5′ TCCAGCCTGTCCAGATGGTGTGTGAGTTTTGTCACAA 3′

The product of this reaction was mixed with primer RMA-76, whichannealed to the amplified product of the first reaction, and the mixturewas amplified with primers KBA-γ2 and RMAK-2.

RMA-76 (SEQ ID NO: 79) 5′ CTAACTCGAGAGTACTGTATGCATGGTTCGAGATGAACAAAGATTCTGAGGCTGCAGCTCCAGCCTGTCCAGATGG 3′

RMAK-2 (SEQ ID NO: 80) 5′ CTAACTCGAGAGTACTGTAT 3′

The PCR product contained a portion of the Fd constant region linkedin-frame to the RMA gene segment. The product also contained a ScaIrestriction site useful for in-frame fusion to a protein such asgelonin, and an XhoI site for subsequent cloning.

This PCR product was cut with SauI and XhoI and ligated adjacent to theremainder of the Fd gene to generate pSH4.

For assembly of the gene fusion vector containing the Fd::RMA::Gelonin,kappa genes, pSH4 was cut with SauI and ScaI and the Fd::RMA segment waspurified. Plasmid PING3731 was cut with SmaI and XhoI and the 760 bp DNAfragment containing the gelonin gene was purified, and pING4000 was cutwith SauI and XhoI and the vector was purified. The product of theligation of these fragments, pING4408, contained the Fd::RMA::Geloninand kappa genes.

(iv) kaDpa::RMA::Gelonin (Fd)

A gelonin gene fusion to the 3′-end of the H65 kappa chain with the 21amino acid RMA linker sequence was assembled in a three piece ligationfrom plasmids pSH6, pING4408 (see the foregoing paragraph) and pING3713.

Plasmid pSH6 contains a kappa gene linked in-frame to the RMA linkersequence. The RMA gene segment was linked to the 3′-end of kappa byoverlap extension PCR as follows. The 3′-end (constant region) of thekappa gene was amplified by PCR from a source plasmid with the primersKBA-K2 and RMAK-1.

RMAK-1 (SEQ ID NO: 81) 5′ TCCAGCCTGTCCAGATGGACACTCTCCCCTGTTGAA 3′

KBA-K2 (SEQ ID NO: 82) 5′ GTACAGTGGAAGGTGGAT 3′

The product of this reaction was mixed with primer RMA-76 (SEQ ID NO:81), which annealed to the amplified product of the first reaction, andthe mixture was amplified with primers KBA-K2 and RMAK-2. The PCRproduct contained a portion of the kappa constant region linked in-frameto the RMA gene segment. The product also contained a ScaI restrictionsite useful for in-frame fusion to a protein such as gelonin, and anXhoI site for subsequent cloning. This PCR product was cut with SstI andXhoI and ligated adjacent to the remainder of the kappa gene to generatepSH6.

For assembly of the gene fusion vector containing thekappa::RMA::Gelonin and Fd genes, pSH6 was cut with HindIII and PstI andthe DNA fragment containing the kappa constant region and a portion ofthe RMA linker (the PstI RMA linker segment contains a PstI site)segment was purified. Plasmid pING4408 was cut with PstI and SalI andthe DNA fragment containing a segment of the RMA linker, the geloningene and a portion of the tetracycline resistance gene in the vectorsegment was purified. pING3713 was cut with SalI and HindIII and thevector was purified. The product of the ligation of these threefragments, pING4410, contained the kappa::RMA::Gelonin and Fd genes.

B. Fusions of Gelonin at the Amino-Terminus of Antibody Genes

(i) Gelonin::SLT::Fd′ (kappa)

A gelonin gene fusion to the 5′-end of the H65 Fd′ chain with a 25 aminoacid SLT linker sequence (20 amino acids from SLT bounded by cysteineresidues, plus five amino acids introduced to facilitate cloning) wasassembled in a three piece ligation from plasmids pING3748, pING3217,and a PCR fragment encoding the H65 heavy chain variable region (V_(H))gene segment which is the variable region of the Fd′ gene in pING3217.Plasmid pING3748 contains the gelonin gene linked in-frame to the SLTlinker sequence, and pING3217 contains the H65 Fd′ and kappa genes in adicistronic transcription unit.

Plasmid pING3825 (see Example 2) was amplified with PCR primersgelo3′-Eag and gelo-9 to introduce an EagI restriction site at the3′-end of the gelonin gene by PCR mutagenesis.

gelo3′-Eag (SEQ ID NO: 83)

5′ CATGCGGCCGATTTAGGATCTTTATCGACGA 3′

The PCR product was cut with BclI and EagI and the 56 bp DNA fragmentwas purified. Plasmid pING3713 was cut with EagI and XhoI, and the 77 bpDNA fragment containing the SLT linker was purified. The 56 bp BclI toEagI fragment and the 77 bp EagI to XhoI fragment were ligated intopING3825 which had been digested with BclI and XhoI to generate pING3748which contains the gelonin gene linked in-frame to the SLT linkersequence.

For assembly of the gene fusion vector containing the Gelonin::SLT::Fd′and kappa genes, the H65 V_(H) was amplified by PCR from pING3217 withprimers H65-G1 and H65-G2, and the product was treated with T4polymerase followed by digestion with NdeI.

H65-G1 (SEQ ID NO: 84) 5′ AACATCCAGTTGGTGCAGTCTG 3′

H65-G2 (SEQ ID NO: 85) 5′ GAGGAGACGGTGACCGTGGT 3′

The 176 bp fragment containing the 5′-end of the H65 heavy chainV-region was purified. Concurrently, pING3217 was digested with NdeI andXhoI, and the 1307 bp DNA fragment containing a portion of the Fd′ geneand all of the kappa gene was purified. The two fragments were ligatedto pING3748 which had been digested with ScaI and XhoI in a three pieceligation yielding pING3754 (ATCC 69102), which contains theGelonin::SLT::Fd′ and kappa genes.

(ii) Gelonin::SLT::kappa (Fd′)

A gelonin gene fusion to the 5′-end of the H65 kappa chain with the 25amino acid SLT linker sequence was assembled in a three piece ligationfrom plasmids pING3748 (see the foregoing section), pING4000, and a PCRfragment encoding the H65 light chain variable region (V_(L)) genesegment.

For assembly of the gene fusion vector containing theGelonin::SLT::kappa and Fd′ genes, an H65 V_(L) fragment was amplifiedby PCR from pING3217 with primers H65-K1 and JK1-HindIII, and theproduct was treated with T4 polymerase followed by digestion with HindIII.

H65-K1 (SEQ ID NO: 86) 5′ GACATCAAGATGACCCAGT 3′

JK1-HindIII (SEQ ID NO: 87) 5′ GTTTGATTTCAAGCTTGGTGC 3′

The 306 bp fragment containing the light chain V-region was purified.Concurrently, pING4000 was digested with HindIII and XhoI, and the 1179bp DNA fragment containing the kappa constant region and all of the Fd′gene was purified. The two fragments were ligated to pING3748 which hadbeen digested with ScaI and XhoI in a three piece ligation yieldingpING3757, which contains the Gelonin::SLT::kappa and Fd genes.

(iii) Gelonin::RMA::Fd′ (kappa)

A gelonin gene fusion to the 5′-end of the H65 Fd′ chain with the 24amino acid RMA linker sequence (20 amino acids from RMA, plus 4 aminoacids introduced to facilitate cloning) was assembled in a three pieceligation from plasmids pING3755, pING3217 and a PCR fragment encodingthe H65 V_(H) gene segment. Plasmid pING3755 contains the gelonin genelinked in-frame to the RMA linker sequence, and pING3217 contains theH65 Fd′ and kappa genes in a dicistronic transcription unit.

Plasmid pING3755 was assembled to contain the gelonin gene linked to theRMA linker gene segment. The RMA linker gene segment was amplified byPCR from pSH4 with primers RMA-EagI and HINDIII-2.

RMA-EagI (SEQ ID NO: 88) 5′ ACTTCGGCCGCACCATCTGGACAGGCTGGAG 3′

HINDIII-2 (SEQ ID NO: 44) 5′ CGTTAGCAATTTAACTGTGAT 3′

The 198 bp PCR product was cut with EagI and HindIII, and the resulting153 bp DNA fragment was purified. This RMA gene segment was clonedadjacent to gelonin using an PstI to EagI fragment from pING3748 and thePstI to HindIII vector fragment from pING3825. The product of this threepiece ligation was pING3755.

For assembly of the gene fusion vector containing the Gelonin::RMA::Fd′,kappa genes, the H65 V_(H) was amplified by PCR from pING3217 withprimers H65-G1 (SEQ ID NO: 84) and H65-G2 (SEQ ID NO: 85), and theproduct was treated with T4 polymerase followed by digestion with NdeI.The 186 bp fragment containing the 5′-end of the heavy chain V-regionwas purified. Concurrently, pING3217 was digested with NdeI and XhoI,and the 1307 bp DNA fragment containing a portion of the Fd′ gene andall of the kappa gene was purified. These two fragments were ligated topING3755 which had been digested with ScaI and XhoI in a three pieceligation yielding pING3759 (ATCC 69104), which contains theGelonin::RMA::Fd′ and kappa genes.

(iv) Gelonin::RMA::kappa (Fd′)

A gelonin gene fusion to the 5′-end of the H65 kappa chain with the 24amino acid RMA linker sequence was assembled in a three piece ligationfrom plasmids pING3755, pING4000, and a PCR fragment encoding the H65V_(L) gene segment.

For assembly of the gene fusion vector containing theGelonin::RMA::kappa and Fd′ genes, an H65 V_(L) segment was amplified byPCR from pING3217 with primers H65K-1 (SEQ ID NO: 86) and JK1-HindIII,and the product was treated with T4 polymerase followed by digestionwith HindIII. The 306 bp fragment containing the 5′-end of the lightchain V-region was purified. Concurrently, pING4000 was digested withHindIII and XhoI, and the 1179 bp DNA fragment containing the kappaconstant region and all of the Fd′ gene was purified. These twofragments were ligated to pING3755 which had been digested with ScaI andXhoI in a three piece ligation yielding pING3758 (ATCC 69103), whichcontains the Gelonin::RMA::kappa and Fd′ genes.

C. Direct Fusions of Gelonin at the Amino Terminus of Antibody Genes

(i) Gelonin::Fd′ (Kappa)

A direct gelonin gene fusion was constructed from pING3754. pING3754 wasdigested with BgIII and XhoI and the vector segment was purified.Concurrently, pING3754 was digested with EagI, treated with T4polymerase, cut with BglII, and the gelonin gene segment was purified.pING3754 was also cut with FspI and XhoI, and the Fd and kappa genesegment was purified. These fragments were assembled in a three-pieceligation to generate pING3334, which contains a direct gene fusion ofgelonin to Fd′ in association with a kappa gene.

EXAMPLE 15

Preparation of he3 Fab and Gelonin he3Fab Immunofusions

The sections below detail the construction of human-engineering he3Fabprotein and immunofusions of gelonin to he3 Fd and kappa chains.

A. he3-Fab Expression Plasmids

The he3 heavy chain V-region was PCR-amplified from plasmid pING4621(pING4621 is fully described above in Example 5 above), with primersH65-G3, GAGATCCAGTTGGTGCAGTCTG (SEQ ID NO: 116) and H65G2 (SEQ ID NO:85). Amplification was carried at using vent polymerase (New EnglandBiolabs) for 25 cycles, including a 94° C. denaturation for 1 minute,annealing at 50° C. for 2 minutes, and polymerization for 3 minutes at72° C. The PCR product was treated with polynucleotide kinase anddigested with BstEII and the V-region DNA was purified. The purified DNAfragment was then ligated into pIC100, which had been digested withSstI, treated with T4 polymerase, and cut with BstEII. The resultingfragment was then ligated with the BstEII fragment from pING3218(containing Fab′ genes) to make pING4623 which contained the he3 Fd genelinked to the pelB leader sequence.

The he3 kappa V-region was next assembled as described above in Example5 and in co-owned, co-pending U.S. patent application Ser. No.07/808,464, incorporated by reference herein, using six oligonucleotideprimers,

           $H65k-1, AGT CGT CGA CAC GAT GGA CAT GAG GAC CCC (SEQ ID NO:117) TGC TCA GTT TCT TGG CAT CCT CCT ACT CTG GTT TCC AGG TAT CAA ATG TGACAT CCA GAT GAC TCA GT;             HUH-K6, TCA CTT GCC GGG CGA ATC AGGACA TTA ATA (SEQ ID NO: 118) GCT ATT TAA GCT GGT TCC AGC AGA AAC CAG GGAAAG CTC CTA AGA CCC T;             HUH-K7, TGA CTC GCC CGG CAA GTG ATAGTG ACT CTG (SEQ ID NO: 119) TCT CCT ACA GAT GCA GAC AGG GAA GAT GGA GACTGA GTC ATC TGG ATG TC;             HUH-K8, GAT CCA CTG CCA CTG AAC CTTGAT GGG ACC (SEQ ID NO: 120) CCA GAT TCC AAT CTG TTT GCA CGA TAG ATC AGGGTC TTA GGA GCT TTC C;             HUH-K4, GGT TCA GTG GCA GTG GAT CTGGGA CAG ATT (SEQ ID NO: 121) ATA CTC TCA CCA TCA GCA GCC TGC AAT ATG AAGATT TTG GAA TTT ATT ATT G; and             HUH-K5, GTT TGA TTT CAA GCTTGG TGC CTC CAC CGA (SEQ ID NO: 122) ACG TCC ACG GAG ACT CAT CAT ACT GTTGAC AAT AAT AAA TTC CAA AAT CTT C and amplified with primers HUK-7 (SEQID NO: 92) and JK1- HindIII (SEQ ID NO: 87).

The resulting PCR product was treated with T4 polymerase, digested withHindIII, and purified. The purified fragment was then cloned intopIC100, which had first been cut with SstI, treated with T4 polymerase,and digested with XhoI, along with the 353 bp HindIII-XhoI fragmentencoding the kappa constant region from pING3217. The resulting plasmidwas pING4627 which contains the he3 kappa sequence linked in frame tothe pelB leader.

Plasmid pING4628, containing the pelB-linked he3 kappa and Fd genesunder transcriptional control of the araB promoter, was assembled frompING4623 and pING4627 as follows.

An expression vector for unrelated kappa and Fd genes, pNRX-2, was firstcut with SauI and EcoRI, leaving a vector fragment which contains allthe features relevant to plasmid replication, a tetracycline resistancemarker, araB transcriptional control, and the 3′ end of the Fd constantregion. [Plasmid pNRX-2 comprises an EcoRI to XhoI DNA segment from pING3104 (described in WO 90/02569, incorporated by reference herein). Thatsegment contains the replication, resistance and transcription controlfeatures of pING3104 and is joined to an XhoI to SauI DNA segment frompING1444 (described in WO 89/00999, incorporated by reference herein)which contains the 3′ end of an Fd constant region.] Next pING4623 wascut with PstI, treated with T4 polymerase, digested with SauI and thepelB::Fd gene segment was then isolated. Plasmid pING4627 was cut withXhoI, treated with T4 polymerase, cut with EcoRI and ligated to thepelB::Fd gene segment and the pNRX-2 vector fragment to generate thehe3-Fab expression vector pING4628. That plasmid contains two XhoIsites, one located between the kappa and Fd genes, and another 4 bpdownstream of the termination codon for the Fd gene.

A vector, pING4633, which lacks the XhoI site between the kappa and Fdgenes was constructed. To assemble pING4633, pING4623 was cut withEcoRI, treated with T4 polymerase, digested with SauI. The pelB::kappagene segment was then isolated and purified. The pNRX-2 vector fragmentand the pelB::Fd gene segment were then ligated to the purifiedpelB::kappa gene segment to form pING4633.

Both pING4633 and pING4628 are bacterial expression vectors for he3-Faband each comprises the he3 Fd and Kappa genes which are expressed as adicistronic message upon induction of the host cell with L-arabinose.Moreover, pING4628 contains two XhoI restriction sites, one located 4 bppast the Fd termination codon and one in the intergenic region betweenthe 3′ end of the Kappa gene and the 5′ end of the Fd gene. PlasmidpING4633 lacks the XhoI site in the intergenic region.

B. Purification of he3Fab

Plasmids pING4628 and pING4633 were transformed into E. coli E104.Bacterial cultures were induced with arabinose and cell-free supernatantcomprising the he3Fab was concentrated and filtered into 20 mm HEPES, pH6.8. The sample was then loaded onto a CM Spheradex column (2.5×3 cm),equilibrated in 20 mM HEPEs, 1.5 mM NaCl, pH 6.8. The column was washedwith the same buffer and eluted with 20 mm HEPES, 27 mM NaCl, pH 6.8.The eluate was split into 2 aliquots and each was loaded onto and elutedfrom a protein G (Bioprocessing) column (2.5×2.5 cm) separately. Theprotein G column was equilibrated in 20 mM HEPES, 75 MM NaCl, pH 6.8 andthe sample was eluted with 100 mM glycine, 100 mM NaCl, pH 3.0. The twoeluates were combined and diluted two times with 20 mM HEPES, 3 Mammonium sulfate, pH 6.8. The diluted eluates were loaded onto phenylsepharose high substitution Fast Flow (Pharmacia) column (2.5×3.3 cm),equilibrated n 20 mM HEPES, 1.5 M ammonium sulfate, pH 6.8. The columnwas then eluted with 20 mM HEPES, 0.6 M ammonium sulfate, pH 6.8.

C. Gelonin::RMA::he3Kappa, he3Fd Fusions

Other genetic constructs were assembled which included a naturalsequence gelonin gene fused to an he3-Fab via a linker.

A fusion comprising Gelonin::RMA::he3Kappa, Fd was assembled from DNAfrom plasmids pING3755, pING4633, and pING4628. Both pING4633 andpING4628 were assembled in a series of steps whereby the he3 heavy andlight V-regions were individually linked in-frame to the pelB leader.The heavy and light V-regions were then placed together in a dicistronicexpression vector under the control of the araB promoter in a bacterialexpression vector.

Assembly of the Gelonin::RMA::he3Kappa, he3Fd fusions was accomplishedby constructing three DNA fragments from plasmids pING3755, pING4633,and pING4628. The first such fragment was made by digesting pING3755with ScaI and XhoI which excises the 4 bp between those sites. Theresulting vector fragment was purified. The second fragment for use inconstructing the above fusions was obtained from plasmid pING4633, whichwas cut with AseI (which cuts in V_(L)) and XhoI and the resulting 1404bp fragment, containing the 3′ end segment of the Kappa and Fd genes,was purified. The third fragment, comprising the 5′ end of the Kappavariable region coding sequence, was prepared from the PCR amplifiedV_(L) gene contained in pING4628 using the oligonucleotide primers,Huk-7 and jk1-HindIII. The resulting 322 bp PCR-amplified V_(L) fragmentwas treated with T4 polymerase, digested with AseI, and the 86 bpfragments containing the 5′ end of V_(L) was purified. The threefragment produced above were ligated together to form pING3764. The DNAsequence of the PCR amplified V-region was verified by direct DNAsequencing of pING3764.

D. Gelonin::SLT::he3Kappa, he3Fd Fusion

A Gelonin::SLT::he3Kappa, he3Fd fusion was constructed by ligating thepING4633 and pING4628 fragments described in section A immediately abovewith a fragment produced from pING3748 which contains Gelonin::SLT. ThepING3748 fragment was produced using ScaI and XhoI as describedimmediately above for pING3755. The resulting vector was designatedpING3763.

E. Construction of Expression Vector Containing Gelonin::SLT::he3Fd,he3kappa Fusions

An expression vector containing the Gelonin::SLT::he3Fd, he3kappa fusionwas constructed in two steps form DNA segments from plasmids pING3825,pING4628, pING4639, pING3217 [described in Better, et al., Proc. Natl.Acad. Sci. (USA), 90:457-461 (1993), incorporated by reference herein],and pING4627. pING3825 was digested with NcoI and XhoI, generating a 654bp fragment containing the 3′ end of the gelonin gene and a fragmentcontaining the 5′ end of the gelonin gene which were purified. Next,pING4639 was digested with NcoI and NdeI and the 903 bp fragmentcontaining the 3′ end of the Gelonin gene, the SLT linker, and the 5′end of V_(E) which resulted was purified. Finally, pING4628 was cut withNdeI and XhoI, resulting in a 523 bp fragment containing the 3′ end ofthe Fd gene which was purified. The three fragments were then ligated toform plasmid pING3765 which contains a gene encoding agelonin::SLT::he3Fd fusion.

Three vector fragments were used to assemble the final expression vector(containing the gelonin::SLT::he3Fd and he3 kappa segments). PlasmidpING3765 was digested with XhoI, treated with T4 polymerase, cut withNheI (which releases a 265 bp fragment encoding the tetracyclineresistent marker), and the resulting vector fragment was purified.Plasmid pING4627, which contains the he3Kappa gene linked in-frame tothe pelB leader was used for the construction of pING4628. PlasmidpING4627 was cut with PstI, treated with T4 polymerase, and furtherdigested with SstI. The resulting 726 bp fragment, containing the Kappagene (except 40 bp at the 3′ end) was purified. Plasmid pING3217 wasthen cut with SstI and NheI, resulting in a 318 bp fragment containingthe 3′ end of the Kappa gene and downstream portion, including a portionof the tetracycline resistance gene, which was purified. Ligation of theforegoing three fragments produced the final expression vector,pING3767.

F. Construction of Expression Vector Containing Gelonin::RMA::he3FdFusions

Gelonin::RMA:he3Fd, he3Kappa fusion expression vectors was constructedin two steps from plasmids pING3825, pING4628, pING3217, and pING4627.The cloning scheme used was identical to that used to generate pING3767except that pING4638 was substituted for pING4639. Plasmid pING4638differs from pING4639 as described below in Example 16. The intermediatevector encoding the Gelonin::RMA::Fd fusion was designated pING3766 andthe final expression vector was designated pING3768.

EXAMPLE 16

Gelonin-Single Chain Antibody Fusions

The natural sequence gelonin gene was also fused to a single chain formof the human engineered he3 H65 variable region. The gelonin gene waspositioned at either the N-terminus or the C-terminus of the fusion geneand the SLT or RMA linker peptide was positioned between the gelonin andantibody domains to allow intracellular processing of the fusion proteinwith subsequent cytosolic release of gelonin.

A. Construction of Gel:: MA::SCA(V_(L)-V_(H)), Gel::SLT::SCA(V_(L)-V_(H)), Gel::RMA:;SCA(V_(H)-V_(L)), andGel::SLT::SCA(V_(H)-V_(L))

A single chain antibody (SCA) form of the he3 H65 variable domain wasassembled from previously constructed genes. This SCA segment consistedof the entire V and J region of the one chain (heavy or light) linked tothe entire V and J segment of the other chain (heavy or light) via a 15amino acid flexible peptide: [(Gly)₄ Ser]₃. This peptide is identical tothat described in Huston et al., Proc. Natl. Acad. Sci. USA,85:5879-5883 (1988); Glockshuber et al., Biochemistry, 29:1362-1367(1990); and Cheadle et al., Molecular Immunol., 29:21-30 (1992). The SCAwas assembled in two orientations:V-J_(kappa)::[(Gly)₄Ser]₃::V-J_(Gamma) andV-J_(Gamma)::[(Gly)₄Ser]₃::V-J_(kappa). Each SCA segment was assembledand subsequently fused to gelonin.

For assembly of the SCA segment V-J_(kappa)::[(Gly)₄Ser]₃:: V-J_(Gamma),primers HUK-7 and SCFV-1 were used to amplify a 352 bp DNA fragmentcontaining the he3 V/J kappa sequences from pING4627 by PCR in areaction containing 10 mM KCl, 20 mM TRIS pH 8.8, 10 mM (NH₄)₂SO₂, 2 mMMgSO₄, 0.1% Triton X-100, 100 ng/ml BSA, 200 uM of each dNTP, and 2Units of Vent polymerase (New England Biolabs, Beverley, Mass.) in atotal volume of 100 μl.

SCFV-1 (SEQ ID NO:91) 5′ CGGACCCACCTCCACCAGATCCACCGCCACCTTTCATCTCAAGCTTGGTGC 3′

HUK-7 (SEQ ID NO: 92) 5′ GACATCCAGATGACTCAGT 3′

concurrently, primers SCFV-2 and SCFV-3 were used to amplify a he3 heavychain V/J gamma segment from pING4623, generating a 400 bp fragment.

SCFV-2 (SEQ ID NO: 93) 5′ GGTGGAGGTGGGTCCGGAGGTGGAGGATCTGAGATCCAGTTGGTGCAGT 3′

SCFV-3 (SEQ ID NO: 94) 5′ TGTACTCGAGCCCATCATGAGGAGACGGTGACCGT 3′

The products from these reactions were mixed and amplified with theoutside primers HUK-7 and SCFV-3. The product of this reaction wastreated with T4 polymerase and then cut with XhoI. The resulting 728 bpfragment was then purified by electrophoresis on an agarose gel. Thisfragment was ligated into the vectors pING3755 and pING3748 (see Example10), each digested with ScaI and XhoI. The resulting vectors pING4637and pING4412 contain the Gelonin::RMA::SCAV-J_(kappa)::[(Gly)₄Ser]₃::V-J_(Gamma) and Gelonin::SLT::SCAV-J_(kappa)::[(Gly)₄Ser]₃::V-J_(Gamma) fusion genes, respectively. The728 bp fragment was also ligated into pIC100 previously digested withSstI, treated with T4 polymerase and digested with XhoI, to generatepING4635. This plasmid contains the pelB leader sequence linked in-frameto the V-J_(kappa)::[(Gly)₄Ser]₃::V-JJ_(gamma) gene. The pelB::SCA genein pING4635 was excised as an EcoRI-XhoI restriction fragment and clonedinto the bacterial expression vector to generate pING4640.

Similarly, the SCA V-J_(Gamma)::[(Gly)₄Ser]₃::V-J_(kappa) was assembledby amplification of pING4627 with primers SCFV-5 and SCFV-6 generating a367 bp fragment containing he3 V/J kappa sequences,

SCFV-5 (SEQ ID NO: 95) 5 GGTGGAGGTGGGTCCGGAGGTGGAGGATCTGACATCCAGATGACTCAGT 3′

SCFV-6 (SEQ ID NO: 96) 5′ TGTACTCGAGCCCATCATTTCATCTCAAGCTTGGTGC 3′

and pING4623 with primers H65-G3 and SCFV-4 generating a 385 bp fragmentcontaining he3 gamma V/J sequences by PCR with Vent polymerase.

H65-G3 (SEQ ID NO: 97) 5′ GAGATCCAGTTGGTGCAGTCTG 3′

SCFV-4 (SEQ ID NO: 98) 5′ CGGACCCACCTCCACCAGATCCACCGCCACCTGAGGAGACGGTGACCGT 3′

The products from these reactions were mixed and amplified with H65-G3and SCFV-6. The 737 bp product was treated with T4 polymerase and cutwith XhoI. Ligation into pING3755 and pING3748 (digested with ScaI andXhoI) resulted in assembly of the Gelonin::RMA::SCAV-J_(Gamma)::[(Gly)₄Ser]₃::V-J_(kappa) gene fusion in pING4638 andGelonin::SLT::SCA V-J_(Gamma)::[(Gly)₄Ser]₃::V-J_(kappa) gene fusion inpING4639, respectively.

The vectors pING4637, pING4412, pING4638 and pING4639 were eachtransformed into E. coli strain E104 and induced with arabinose. Proteinproducts of the predicted molecular weight were identified by Westernblot with gelonin-specific antibodies.

B. Construction of SCA(V_(L)-V_(H)::SLT::Gelonin Vectors

The expression vector containing SCA (V_(L-V) _(H))::SLT::Geloninfusions was assembled using restriction fragments frompreviously-constructed plasmids pING4640 (containing SCA(V_(L)-V_(H)) )pING4407 (containing Kappa::SLT::Gelonin, Fd), and pING3197. PlasmidpING4640 was first cut with BspHI, filled in with T4 polymerase in thepresence of only dCTP, treated with mung bean nuclease (MBN) to removethe overhang and to generate a blunt end, and cut with EcoRI. Theresulting 849 bp fragment was purified. The SLT-containing fragment frompING4407 was excised by cutting with EagI, blunted with T4 polymerase,cut with XhoI, and the approximately 850 bp fragment which resulted waspurified. The two fragments were ligated together into pING3197, whichhad been treated with EcoRI and XhoI to generate pING4642. The DNAsequence at the BspHI-T4-MBN/EagI junction revealed that two of theexpected codons were missing but that the fusion protein was in frame.

C. Construction of SCA(V_(H)-V_(L))::SLT::Gelonin Vectors

The expression vector containing the SCA(V_(H)-V_(L))::SLT::Geloninfusions was assembled using DNA from plasmids pING4636, (the E. coliexpression vector for SCA(V_(H)-V_(L))) and pING4407. Plasmid pING4636was cut with BstEII and XhoI and the resulting vector fragment waspurified. Concurrently, pINg4636 was used as a template for PCR withprimers SCFV-7, 5′TGATGCGGCCGACATCTCAAGCTTGGTGC (SEQ ID NO: 112) andH65-G13, TGATGCGGCCGACATCTCAAGCTTGGTGC3′ (SEQ ID NO: 113). The amplifiedproduct was digested with EagI and BstEII and the resultingapproximately 380 bp fragment was purified. Plasmid pING4407 was thencut with EagI and XhoI, resulting in an approximately 850 bp fragment,which was purified. The three above fragments were ligated together toproduce pING4643.

D. Construction of SCA(V_(L)-V_(H)::RMA::Gelonin Vectors

Expression vectors containing SCA(_(L)-V_(H))::RMA::Gelonin fusions wereassembled using DNA from pING4640, pING4408 [Example 14A(iii)], andpING3825 (Example 2C). Plasmid pING4640 was cut with SalI and BstEII andthe resulting approximately 700 bp vector fragment (containing thetetracycline resistance matter) was purified. Next, pING3825 wasdigested with NcoI and SalI, resulting in an approximately 1344 bpfragment containing the 3′ end of the gelonin gene and adjacent vectorsequences. That fragment was purified. Plasmid pING4408 was then PCRamplified with oligonucleotide primers, RMA-G35′TCTAGGTCACCGTCTCCTCACCATCTGGACAGGCTGGA3′ (SEQ ID NO: 114), andgelo-10. The resulting PCR product was cut with BstEII and NcoI togenerate an approximately 180 bp fragment containing the 3′ end ofV_(H), RMA, and the 5′ end of the Gelonin gene which was purified. Theabove three fragments were ligated to generate the final expressionvector, pING4644.

E. Construction of SCA (V_(H)-V_(L))::RMA::Gelonin Vectors

Expression vectors containing SCA(V_(H)-V_(L))::RMA::Gelonin wereconstructed using DNA from pING 4636, pING4410, and pING3825. PlasmidpING4636 was digested with SalI and HindIlI and the resulting vectorfragment was purified. Next, pING3825 was cut with NcoI and SalI and the1344 bp fragment which resulted contained the 3′ end of the gelonin geneand adjacent vector sequences encoding tetracycline resistance waspurified. Finally, pING4410 was PCR amplified with primers RMA-G4,5′TTCGAAGCTTGAGATGAAACCATCTGGACAGGCTGGA3′ (SEQ ID NO: 115) and gelo-10.The PCR product was cut with HindIII and NcoI, resulting in a 180 bpfragment containing the 3′ end of V_(L,) RMA, and the 5′ end of Geloninand was purified. The three above fragments were ligated together togenerate the final expression vector, pING4645.

Gelonin::SCA fusions without a cleavable linker may be constructed bydeletion of the SLT linker in pING4412 using the restriction enzymesEagI and FspI. Digestion at these sites and religation of the plasmidresults in an in-frame deletion of the SLT sequence.

EXAMPLE 17

Multivalent Immunofusions

Multivalent forms of the immunofusions may be constructed.

A. Construction of (Gel::RMA::kappa, Fd′)₂ and (Gel::RMA::Fd′, kappa)₂Expression Vectors

Bacterial Fab expression vectors can result in the production of F(ab′)₂if the two cysteine residues from the human IgG1 hinge region areincluded into the carboxyl-terminus of the Fd protein [Better et al.,Proc. Natl. Acad. Sci. USA, 90:457-461 (1993)]. To express a geloninfusion protein that could form a bi-valent structure like an F(ab′)₂,the he3 Fd′ (2C) hinge region (Better et al., supra) was cloned into theexpression vector pING3764 (Example 15C) encoding the fusion proteinGel::RMA::kappa, Fd.

Plasmid pING3764 was cut with XhoI and Bsu36I and the approximately 7500bp fragment containing the immunofusion gene and vector sequences waspurified. Plasmid pING4629, which encodes he3 F(ab′)₂, was also cut withBsu36I and XhoI, and the approximately 200 bp DNA fragment containingthe he3 Fd′ (2C) gene segment was purified. These two DNA fragments wereligated to generate pING3775 encoding (Gel::RMA::kappa, Fd′)₂. Anexpression vector encoding the fusion protein (Gel::RMA::Fd′, kappa)₂was also made.

B. Construction of Vectors Containing Both Gel::RMA::Fd and Gel::RMA::KFusions

In order to construct a plasmid comprising Gel::RMA::Fd and Gel::RMA::kfusions, plasmid pING3764 [described above in Example 15(b)] wasdigested with BsgI and SauI and a 5.7 kb vector fragment containingplasmid replication functions, Gel::RMA::k, and the 3′ end of Fd wasisolated and purified. Plasmid pING3768 [described above in Example15(E)] was digested with SauI and partially digested with PstI and a 1.5kb fragment containing Gel::RMA::Fd was purified. Finally, pING4000[described above in Example 14] was digested with BsgI and PstI,generating a 350 bp fragment containing the 3′ end of the kappa gene.That fragment was purified and the 5.7 kb, 1.5 kb, and 350 kb fragmentsdescribed above were ligated together to form pING3770, containing thegelonin::RMA::k and gelonin:RMA::Fd fusions.

C. Construction of Vectors Containing Both Gel::SLT::Fd and Gel::SLT::kFusions

Plasmid pING3772 contains the above-entitled fusions and was constructedas follows. Plasmid pING3763 [described above in Example 15(D)] wasdigested with BsgI and SauI and a 5.7 kb fragment containing thereplication functions, the 5′ end of Gel::SLT::k and the 3′ end of Fdwas generated and purified. Next, plasmid pING3767 [described in Example15(D) above] was digested with SauI and PstI, generating a 1.5 kbfragment containing the 5′ end of the gel::SLT::Fd fusion. That fragmentwas purified and pING4000 [described in Example 14 above] was digestedwith BsgI and PstI. The resulting 350 bp fragment was purified and theabove-described 5.7 kb, 1.5 kb, and 350 bp fragments were ligated toform pING3772.

D. Expression of Multivalent Fusions

Both pING3770 and pING3772 were transformed into E. coli (E104) cells bytechniques known to those of ordinary skill in the art and induced witharabinose. Concentrated supernatants from the transformed call cultureswere analyzed by Western blot analysis with rabbit anti-geloninantiserum. Transformants from both plasmids generated a reactive band onthe gel at the size expected for a Fab molecule carrying two gelonins(approximately 105 kD). These results are consistent with the productionof fusion proteins comprising monovalent Fab, with both Fd and kappachains separately fused to gelonin.

E. coli strains containing plasmids pING3775, pING3770 and pING3772 weregrown in fermenters and the fusion protein products were purified. The(Gel::RMA::kappa,Fd′)₂ expressed from pING3775 was purified as describedin Better et al., supra.

EXAMPLE 18

Construction of Expression Vectors Encoding Immunofusions withoutLinkers

Expression vectors encoding direct fusions of gelonin and dicistroniche3 Fab protein or single chain antibody were constructed as follows.

A. V_(H)V_(L)::Gel

Plasmid pING4642 (Example 16B) which encodes the V_(L)V_(H)::SLT::Gelfusion protein was cut with FspI and NcoI, and the approximately 100 bpDNA fragment containing the 5′-end of the gelonin gene was purified.Plasmid pING4643 (Example 16C), which encodes the V_(H)V_(L)::SLT::Gelfusion protein, was cut with EagI, treated with T4 polymerase and cutwith PstI. The approximately 850 bp DNA fragment encoding the V_(H)V_(L)gene segment was purified. The DNA fragments from pING4642 and pING4643were ligated into the vector DNA fragment from pING4644 (Example 16D)that had been cut with PstI and NcoI to generate pING3781, which encodesthe V_(H)V_(L)::Gel direct gene fusion.

B. V_(L)V_(H)::Gel

Plasmid pING4640 which encodes the he3 SCA gene V_(L)V_(H) was cut withBspHI, treated with T4 polymerase in the presence of the nucleotide dCTPonly, treated with mung bean nuclease to remove the remaining 5′overhang, and then cut with EcoRI. The approximately 800 bp DNA fragmentcontaining the he3 V_(L)V_(H) gene was then purified on an agarose gel.

Plasmid pING3781 which encodes the direct fusion V_(H)V_(L)::Gel wasdigested with EagI, treated with T4 polymerase, and then digested withXhoI. The approximately 800 bp DNA fragment encoding the gelonin genewas then purified on an agarose gel. The two DNA fragments from pING4640and pING3781 were ligated into the vector DNA from pING3767 which hadbeen digested EcoRI and XhoI and purified on an agaraose gel. Theresultant plasmid, pING3348, encoded the V_(L)V_(H)::Gel fusion protein.The DNA sequence at the fusion junction was verified by direct DNAsequencing.

C. Gel::V_(H)V_(L)

The plasmid pING3755 [Example 14B(iii)], which contains the gelonin genewith an engineered EagI site at its 3′-end, was cut with EagI, treatedwith T4 polymerase, and digested with NcoI. The approximately 650 bp DNAfragment containing the 3′-end of the gelonin gene was purified on anagarose gel. The plasmid pING4639 (Example 16A) encoding the fusionGel::SLT::V_(H)V_(L) was cut with XhoI and then partially digested withFspI. The approximately 730 bp DNA fragment containing all of the he3V_(H)V_(L) gene was then purified in an agarose gel (a single FspIrestriction site occurs in the V_(H) gene segment, and the purified he3V_(H)V_(L) gene was separated from the incomplete gene segment which wasapproximately 660 bp). The two DNA fragments from pING3755 and pING4639were ligated into the vector pING3825 that had been digested with NcoIplus XhoI and purified on an agarose gel. The plasmid pING3350 wasgenerated which encoded the Gel::V_(H)V_(L) fusion protein. The DNAsequence at the fusion junction was verified by direct DNA sequencing.

D. Gel::V_(L)V_(H)

Plasmid pING3336 which encodes the he3 V_(L)V_(H) single chain antibodygene was cut with SstI and AseI, and the approximately 5500 bp DNAfragment containing the 3′-end of V_(L)V_(H) and downstream vectorsequences was purified. (pING3336 is identical to pING4640 except thatthe V_(L)V_(H) gene encodes six histidine residues in frame at thecarboxyl-terminus). Plasmid PING4627 (Example 15A) served as a substratefor PCR amplification of the V_(H) gene segment. Plasmid pING4627 wasamplified with the two oligonucleotide primers HUK-7 (SEQ ID NO: 92) andJK1-HindIII (SEQ ID NO: 87), the resultant product was treated with T4polymerase and cut with Asel, and the 86 bp DNA fragment containing the5′-end of the VL was purified. The DNa fragments from pING3336 andpING4627 were ligated to the approximately 2350 bp DNA fragment ofpING3755 generated by digestion with EagI, treatment with T4 polymeraseand subsequent digestion with SstI. The resultant vector containing theGel::V_(L)V_(H) gene fusion was named pING4652. The DNA sequence ofpING4652 was verified at ligation juctions.

E. Gel::kappa, Fd

The direct gene fusion which encodes Gel::kappa, Fd was also assembledfrom DNA segments from three plasmids. Plasmid pING3764 (Example 15C)was digested with HindIII and XhoI, and the approximately 1200 bp DNAfragment encoding the 3′-end of the kappa gene and the Fd gene waspurified. Plasmid pING4652, which encodes a direct gene fusion ofgelonin to the he3 SCA gene V_(L)V_(H), was cut with BglII and HindIII,and the approximately 850 bp DNA fragment encoding the 3′-end of thegelonin gene and the V_(L) region of kappa was purified. The DNAfragments from pING3764 and pING4652 were ligated into the vectorfragment from pING3825 (Example 2C) that had been digested with BglIIand XhoI to generate pING3784 encoding Gel::kappa, Fd.

F. Gel::Fd, kappa

Plasmid pING3768 (Example 15F), which encodes the fusion proteinGel::RMA::Fd, kappa, was cut with NdeI and NheI, and the DNA segmentcontaining the majority of the he3 Fd gene, the he3 kappa gene and aportion of the tetracycline resistance gene of the vector was purified.Plasmid pING3350, which is described in section C above, was cut withNdeI and PstI, and the DNA fragment containing the 5′-end of the he3 Fdgene linked to the gelonin gene was purified. The DNA fragments frompING3350 and pING3768 were ligated into the vector fragment frompING4633 (Example 16D) that had been cut with Nhel and Pstl to generatepING3789. Plasmid pING3789 encodes the fusion protein Gel::Fd, kappa.

EXAMPLE 19

Alternative Cathepsin Cleavable Linkers

The segment of rabbit muscle aldolase chosen for the RMA linkerdescribed herein is known to contain peptide sequences susceptible todigestion with cathepsins. Other cathepsin-cleavable protein segmentsare effective targets for intracellular cleavage, and two particularamino acid sequences were included as cleavable linkers in additionalimmunofusions of the invention. These are the amino acid sequenceKPAKFFRL (SEQ ID NO: 141 (“CCF”) and KPAKFLRL (SEQ ID NO: 142) (“CCL”).Two oligonucleotides were synthesized that encode these peptidesegments. Degeneracy was introduced at one nucleotide position in eachsynthetic primer to allow the appropriate amino acid to be encoded atthe particular amino acid position in which CCF and CCL differ. The twooligonucleotides 5′-GGCCGCAAAGCCGGCTAAGTTCTT(A/C)CGTCTGAGT-3′ (SEQ IDNO: 143) and 5′-ACTCAGACG(G/T)AAGAACTTAGCCGGCTTTGC-3′ (SEQ ID NO: 144).The oligonucleotide linkers were then used to assemble a family offusion gene expression vectors encoding: Gel::CCL::kappa, Fd;Gel::CCF::kappa, Fd; Gel::CCF::V_(L)V_(H); and Gel::CCL::V_(H)V_(L).

The CCL and CCF linkers were also included in fusion vectors where theantigen-binding domain of the fusion protein was at the N-terminus ofthe fusion to generate expression vectors encoding immunofusions such asV_(L)V_(H)::CCL::Gel.

Several of the fusion proteins with the CCL and CCF linkers were testedfor cytotoxicity on the T cell lines HSB2 and PBMC and were comparablein activity to the fusion proteins containing the RMA linker.

EXAMPLE 20

Expression and Purification of Gelonin Immunofusions

A. Expression of Gelonin Immunofusions

Each of the gelonin gene fusions whose construction is described inExample 15 was co-expressed with its pair H65 Fab gene inarabinose-induced E. coli strain E104.

Expression products of the gene fusions were detected in the supernatantof induced cultures by ELISA. Typically, a plate was coated withantibody recognizing gelonin. Culture supernatant was applied and boundFab was detected with antibody recognizing human kappa coupled tohorseradish peroxidase. H65 Fab fragment chemically conjugated togelonin was used a standard. Alternative ELISA protocols involvingcoating a plate with antibody recognizing either the kappa or Fd orinvolving a detection step with anti-human Fd rather than anti-humankappa yielded similar results. Only properly assembled fusion proteincontaining gelonin, kappa and Pd was detected by this assay.Unassociated chains were not detected.

The fusion protein produced from induced cultures containing expressionvectors pING4406, 4407, 4408, and 4410 in E. coli E104 accumulated atabout 20-50 ng/ml. The fusion proteins expressed upon induction ofpING3754, 3334, 3758 and 3759 (but not pING3757) were expressed at muchhigher levels, at about 100 to 500 ng/ml. A fusion protein of about70,000 Kd was detected in the concentrated E. coli culture supernatantby immunostaining of Western blots with either anti-human kappa oranti-gelonin antibodies.

The Gelonin::SLT::Fd′ (kappa) fusion protein from pING3754 (ATCC 69102)was purified from induced 10 L fermentation broth. The 10 L fermentationbroth was concentrated and buffer exchanged into 10 mM phosphate bufferat pH 7.0, using an S10Y10 cartridge (Amicon) and a DC10 concentrator.The supernatant was purified by passing the concentrated supernatantthrough a DE52 column (20×5 cm) equilibrated with 10 mM sodium phosphatebuffer at pH 7.0. The flow-through was then further purified andconcentrated by column chromatography on CM52 (5×10 cm) in 10 mMphosphate buffer. A 0-0.2 M linear gradient of NaCl was used to theelute the fusion protein, and fractions containing the fusion proteinwere pooled and loaded onto a Protein G column (1 ml). The fusionprotein was eluted from protein G with 0.2 M sodium citrate, pH 5.5 andthen 0.2 M sodium acetate, pH 4.5, and finally, 0.2 M glycine, pH 2.5.The Gelonin::RMA::Fd′ (kappa) and Gelonin::RMA::kappa (Fd′) fusionsproteins were purified from fermentation broths by similar methodsexcept that the CM52 column step was eliminated, and the DE52 column wasequilibrated with 100 mM sodium phosphate buffer at pH 7.0. The fusionproteins were not purified to homogeneity.

Each of the three purified fusion proteins was then assayed for activityin the RLA assay and for cytotoxicity against the T-cell line HSB2. (Tcells express the CD5 antigen which is recognized by H65 antibody.) TheRLA assay was performed as described in Example 4 and results of theassay are presented below in Table 12.

TABLE 12 Fusion Protein IC50 (pM) rGelonin 11 Gelonin::SLT::Fd (kappa)19 Gelonin::RMA::Fd (kappa) 28 Gelonin::RMA::kappa (Fd) 10

Two fusion proteins were tested in whole cell cytotoxicity assaysperformed as described in Example 6 (Table 13). As shown in Table 13,the fusion proteins were active. Gelonin::SLT::Fd(kappa) killed two Tcell lines, HSB2 and CEM, with respective IC₅₀s 2-fold (HSB2) or 10-fold(CEM) higher than that of the gelonin chemically linked to H65. SeeTable 13 below for results wherein IC₅₀ values were adjusted relative tothe amount of fusion protein in each sample.

TABLE 13 IC₅₀ (pMT) Fusion Protein HSB2 Cells CEM Cellshe3Fab-Gel_(A50(C44)) 165  173 Gelonin::SLT::Fd (kappa) 180 1007Gelonin::RKA::Fd (kappa) 150 NT These fusion protein showed similaractivity on peripheral blood mononuclear cells (data not shown).

B. Purification of Immunofusions

(i) Immunofusions Comprising cH65Fab′

Immunofusions comprising a cH65Fab′ fragment were purified fromcell-free supernatants by passing the supernatant through a CM Spheradex(Sepacor) column (5 cm×3 cm), equilibrated in 10 Mm Na phosphate at pH7.0. Immunofusion proteins bind to the column and are eluted with 10 mMNa phosphate, 200 mM NaCl, pH 7.0. The eluate was diluted two-fold with20 Mm HEPES, 3 M ammonium sulfate, pH 7.6 and loaded onto a phenylsepharose fast flow (Pharmacia) column (2.5×3.5 cm), equilibrated in 20mM HEPES, 1.2 M ammonium sulfate, pH 7.0. The column was next washedwith 20 mM Hepes, 1.2 ammonium sulfate, pH 7.0 and eluted with 20 mMHEPES, 0.9 M ammonium sulfate, pH 7.0. The phenyl sepharose eluate wasconcentrated to a volume of 2-4 ml in an Amicon stirred cell fitted witha YM10 membrane. The concentrated sample was loaded onto an S-200(Pharmacia) column (3.2×38 cm), equilibrated in 10 mm Na phosphate, 150mm NaCl, pH 7.0. The column was run in the same buffer and fractionswere collected. Fractions containing the fusion protein of desiredmolecular weight were combined. For example, by selection of appropriatecolumn fractions, both monovalent (gelonin-Fab′) and bivalent(gelonin₂-F(ab′)₂ forms encoded by pING3758 were purified.

(ii) Immunofusions Comprising he3Fab

Immunofusions comprising he3Fab were purified as in the precedingsection with the exception that the phenyl sepharose column was elutedwith 20 mM HEPES, 1.0 M ammonium sulfate, pH 7.0.

(iii) Immunofusions Comprising SCA

Cell-free supernatant was passed through a CM spheradex column (5×3 cm),equilibrated with 10 mM Na phosphate, pH 7.0. Single-chain antibodybinds to the column which is then washed with 10 mM Na phosphate, 45 mMNaCl, pH 7.0. The fusion protein was then eluted with 10 mM Naphosphate,200 mM NaCl, pH 7.0. The eluate was diluted two-fold with 20 HEPES, 3 Mammonium sulfate, pH 7.0 and loaded onto a butyl sepharose Fast Flow(Pharmacia) column (2.5×4.1 cm) equilibrated in 20 mM HEPES, 1.5 Mammonium sulfate, pH 7.0. The column was then washed with 20 mM HEPES,1.0 M ammonium sulfate, pH 7.0 and eluted with 20 mM HEPES pH 7.0. Thebutyl sepharose eluate was concentrated to a volume of 2-4 ml in anAmicon stirred cell fitted with a YM10 membrane. The concentrated samplewas loaded onto an S-200 (Pharmacia) column (3.2×38 cm) equilibrated in10 mM Na phosphate, 150 mM NaCl, pH 7.0. The column was then run in thesame buffer and the fractions were collected. Some of the fractions wereanalyzed by SDS-PAGE to determine which fractions to pool together forthe final product.

EXAMPLE 21

Activity of Gelonin Immunofusions

A concern in constructing immunofusions comprising any RIP is that thetargeting and enzymatic activities of the components of the fusionprotein may be lost as a result of the fusion. For example, attachmentof an RIP to the amino terminus of an antibody may affect theantigen-binding (complementarity-determining regions) of the antibodyand may also result in steric hinderance at the active site. Similarly,the activity of an RIP may be hindered by attachment of an antibody orantibody portion. For example, RIPs chemically conjugated to antibodiesvia a disulfide bridge are typically inactive in the absence of reducingagents. In order to assess the foregoing in immunofusions of the presentinvention, such proteins were subjected to assays to determine theirenzymatic, binding, and cytotoxic activities.

A. Reticulocyte Lysate Assay

The enzymatic activity of immunofusions comprising gelonin was assayedusing the reticulocyte lysate assay (RLA) describe above. As noted inExample 4, the RLA assay measures the inhibition of protein synthesis ina cell-free system using endogenous globin mRNA from a rabbit red bloodcell lysate. Decreased incorporation of tritiated leucine (³H-Leu) wasmeasured as a function of toxin concentration. Serial log dilutions ofstandard toxin (the 30 kD form of ricin A-chain, abbreviated as RTA 30),native gelonin, recombinant gelonin (rGelonin or rGel) and geloninanalogs were tested over a range of 1 μg/ml to 1 pg/ml. Samples weretested in triplicate, prepared on ice, incubated for 30 minutes at 37°C., and then counted on an Inotec Trace 96 cascade ionization counter.By comparison with an uninhibited sample, the picomolar concentration oftoxin (pM) which corresponds to 50% inhibition of protein synthesis(IC₅₀) was calculated.

Representative data for various immunotoxins of the invention are shownbelow in Table 14.

TABLE 14 Immunotoxin Lot No. IC₅₀ (pM) rGel::RMA::SCA(V_(E)-V_(L))AB1136 12 rGel::RMA::SCA(V_(L)-V_(E)) AB1137 18rGel::SLT::SCA(V_(E)-V_(L)) AB1133 26 rGel::RMA::SCA(V_(L)-V_(E)) AB112433 rGel::RMA::K + Fd′ (cH65Fab′) AB1122 54 rGel::SLT::K + Fd (he3Fab)AB1160 40 rGel::RMA::K + Fd (he3Fab) AB1149 33 rGel::RMA::Fd + K(he3Fab) AB1163 14 rGel::Fd′ + K (cH65Fab′) AB1123 45

Contrary to the expectations discussed above, gelonin immunofusions ofthe invention exhibit enzymatic activity which is comparable to theactivities of native and recombinant gelonin shown in Example 4. Thiswas true for fusions made with either the reducible (SLT) ornon-reducible (RMA) linkers.

B. Binding Activity of Immunofusions

Several immunofusions according to the present invention were assayedfor their ability to compete with labelled antibody for binding toCD5-positive cells. The Kd of the immunofusions was estimated by threedifferent means as shown in Table 15. The first Kd estimation (Kd₁ inTable 15) was obtained by competition with fluorescein-labelled H65 IgGfor binding to MOLT-4X cells (ATCC CRL 1582) according to the procedurereported in Knebel, et al., Cytometry Suppl., 1: 68 (1987), incorporatedby reference herein.

The second Kd measurement (Kd₂ in Table 15) was obtained by Scatchardanalysis of competition of the immunofusion with ¹²⁵I-cH65 IgG forbinding on MOLT-4M cells as follows. A 20 μg aliquot of chimeric H65 IgG(cH65 IgG) was iodinated by exposure to 100 μl lactoperoxidase-glucoseoxidase immobilized beads (Enzymobeads, BioRad), 100 μl of PBS, 1.0 mCiI¹²⁵ (Amersham, IMS30), 50 μl of 55 mM b-D-glucose for 45 minutes at 23°C. The reaction was quenched by the addition of 20 μl of 105 mM sodiummetabisulfite and 120 mM potassium iodine followed by centrifugation for1 minute to pellet the beads. ¹²⁵I-cH65 IgG was purified by gelfiltration using a 7 ml column of sephadex G25, eluted with PBS (137 mMNaCl, 1.47 mM KH₂PO₄, 8.1 mM Na₂HPO₄, 2.68 mM KCl at pH 7.2-7.4) plus0.1% BSA. ¹²⁵I-cH65 IgG recovery and specific activity were determinedby TCA precipitation.

Competitive binding was performed as follows: 100 μl of Molt-4M cellswere washed two times in ice-cold DHB binding buffer (Dubellco'smodified Eagle's medium (Gibco, 320-1965PJ), 1.0% BSA and 10 mM Hepes atpH 7.2-7.4). Cells were resuspended in the same buffer, plated into 96v-bottomed wells (Costar) at 3×10⁵ cells per well and pelleted at 4° C.by centrifugation for 5 min at 1,000 rpm using a Beckman JS 4.2 rotor;50 μl of 2×-concentrated 0.1 nM ¹²⁵I-cH65 IgG in DHB was then added toeach well and competed with 50 μl of 2×-concentrated cH65 IgG in DHB atfinal protein concentrations from 100 nM to 0.0017 nM. Theconcentrations of assayed proteins were determined by measuringabsorbance (A₂₈₀ and using an extinction coefficient of 1.0 for fusionproteins, 1.3 for Fab, and 1.22 for Fab conjugated to gelonin. Also,protein concentrations were determined by BCA assay (Pierce Chemical)with bovine serum albumin as the standard. Binding was allowed toproceed at 4° C. for 5 hrs and was terminated by washing cells threetimes with 200 μl of DHB binding buffer by centrifugation for 5 min. at1,000 rpm. All buffers and operations were at 4° C. Radioactivity wasdetermined by solubilizing cells in 100 μl of 1.0 M NaOH and counting ina Cobra II auto gamma counter (Packard). Data from binding experimentswere analyzed by the weighted nonlinear least squares curve fittingprogram, MacLigand, a Macintosh version of the computer program “Ligand”from Munson, Analyt. Biochem., 107:220 (1980), incorporated by referenceherein.

Finally, the Kd (Kd₃ in the Table) was estimated by examination of theED₅₀ values obtained from separate competition binding assays, performedas described in the previous paragraph. All three measurements are shownin Table 15 below:

TABLE 15 Molecule Type Kd₁ Kd₂ Kd₃ H65 IgG 1.6  ND ND cH65 IgG ND 3.0  2.5 cH65Fab′ 4.0  14.0  ND cH65Fab′-rGel_(A50(C44)) 3.5  13.0  NDrGel::RMA::K + Fd′ (cH65Fab′) 16.0  ND 100  he3Fab 1.20  2.60 NDhe3Fab-rGel_(A50(C44)) 1.10  2.70 ND rGel::RMA::K + Fd′ (he3Fab) 2.60 ND  5.0 rGel::SLT::K + Fd (he3Fab) ND ND 30 SCA(V_(L)-V_(E)) 2.20 ND 30rGel::RMA::SCA(V_(E)-V_(L)) 3.50 ND 20 rGel::RMA::SCA(V_(L)-V_(E)) 4.70ND 30 SCA(V_(L)-V_(E)) ND ND 20 rGel::RMA::SCA(V_(L)-V_(E)) 2.30 ND NDND = not determined

The results presented in Table 15 suggest that Fab and SCA antibodyforms may retain substantial binding activity even when fused to an RIP.

C. Comparative Cytotoxicity Assays

Fusion proteins and immunoconjugates according to the present inventionwere used in a comparative cytoxicity assay. Two types of assays wereconducted, one targeting T cell line HSB2, and the other targetinglectin-activated peripheral blood mononuclear cells (PBMC) according toprocedures in Example 6. The results of the assays are presented belowin Tables 16a, 16b and 16c.

TABLE 16a CYTOTOXIC POTENCIES: CHEMICAL VS. GENE-FUSED CONJUGATES HSB2Fusion IC₅₀, IC₅₀, Immunotoxin Lot # Plasmid pM Toxin pM Toxin N CD5Plus HF002002 —    148 24 8 H65-M- 999 —    68* NA 1 rGel_(A50(C44))cH65-MM-rGel 807 —    183* NA 1 cH65Fab′-M- 941 —    99 6 2rGel_(A50(C44)) He2Fab-M- 970 —    468 195 4 rGel_(A50(C44)) he3Fab-M-1012/1047 —    190 70 12 rGel_(A50(C44)) he3Fab-SMCC- 1086 —  5,9042,442 2 rGel_(A50(C44)) rGel::SLT::Fd′ + AB1095 pING3754    320 25 2K(1)⁺ rGel::SLT::Fd′ + AB1095 pING3754    374* NA 1 K(3)⁺ rGel::SLT::K +AB1147 pING3763    495* NA 1 Fd(he3) rGel::SLT::K + AB1160 pING3763   746* NA 1 Fd(he3) rGel::SLT:: AB1133 pING4639    422 31 5 SCA(Vh-Vl)rGel::SLT:: AB1124 pING4412    776 347 3 SCA(Vl-Vh) rGel::RMA::K +AB1122 pING3758  1,506 1,033 2 Fd′ rGel::RMA::K + AB1141 pING3758 5,833* NA 1 Fd′ rGel::RMA::K + AB1149 pING3764  9,154* NA 1 Fd(he3)rGel::RMA::K + AB1161 pING3764  5,974* NA 1 Fd(he3) rGel::RMA::Fd′ +RF524(1) pING3759  1,955* NA 1 K rGel::RMA::Fd′ + AB1121 pING3759 32,051* NA 1 K rGel::RMA::Fd + AB1163 pING3768  3,256* NA 1 K(he3)rGel::RMA:: AB1136 pING4638  3,687 1,144 6 SCA(Vh-Vl) rGel::RMA:: AB1152pING4638  41,218* NA 1 SCA(Vh-Vl) rGel::RMA:: AB1137 pING4637  11,979*NA 1 SCA(Vl-Vh) rGel::RMA:: AB1164 pING4637  1,146* NA 1 SCA(Vl-Vh)rGel::Fd′ + AB1123 pING3334  6,346* NA 1 K K::RMA::rGel + AB1140pING4410  10,090* NA 1 Fd rGel 1056 —  46,600 34,600 3 B72.3Fab-M- 1057— 129,032* NA 1 rGel_(A50(C44)) *Results represent single values and nota mean value. ⁺rGel::SLT::Fd′ + k(1) and rGel::SLT::Fd′ + k(3) areseparate fractions from the final purification column.

TABLE 16b CYTOTOXIC POTENCIES: CHEMICAL VS. GENE-FUSED CONJUGATES PBMCIC₅₀, IC₅₀, Immunotoxin Lot # pM Toxin pM Toxin SD N CD5 Plus HF0020021,095 1,236 908 18 H65-m- 999 133 133 129 2 rGel_(A50(C44)) cH65-m2-rGel807 143 308 492 8 cFab′-rGel_(A50(C44)) 941 434 405 280 4 He2Fab- 970397 397 146 2 rGel_(A50(C44)) he3Fab- 1012/1047 206 307 274 18rGel_(A50(C44)) he3Fab-smcc- 1086 335 638 538 3 rGel_(A50(C44))rGel::SLT::Fd′ + AB1095 15,840 15,840 15,783 2 K(1)⁺ rGel::SLT;:Fd′ +AB1095 2,350 4,322 4,159 9 K(3)⁺ rGel::SLT::K + AB1147 1,890 1,407 1,0155 Fd(he3) rGel::SLT::K + AB1160 2,910 4,584 5,100 3 Fd(he3) rGel::SLT::AB1133 1,125 1,870 1,637 6 SCA(Vh-Vl) rGel::SLT:: AB1124 2,725 2,815 7434 SCA(Vl-Vh) rGel::RMA::K + AB1122 211 307 250 14 Fd′ rGel::RMA::K +AB1141 4,400 4,041 2,691 4 Fd′ rGel::RMA::K + RF-532 15,000 9,114 8,3253 Fd′ rGel::RMA::K + AB1149 7,124 10,764 14,081 5 Fd(he3) rGel::RMA::K +AB1161 1,854 2,990 3,324 3 Fd(he3) rGel::RMA::Fd′ + RF524(1) 1,760 1,8931,049 5 K rGel::RMA::Fd′ + AB1121 2,090 1,664 1,553 6 K rGel::RMA::Fd +AB1163 854 567 406 2 K(he3) rGel::RMA:: AB1136 393 567 510 7 SCA(Vh-Vl)rGel::RMA:: AB1152 9,650 9,170 6,483 3 SCA(Vh-Vl) rGel::RMA:: AB11374,040 4,554 4,310 7 SCA(Vl-Vh) rGel::RMA:: AB1164 1,598 1,598 1,144 2SCA(Vl-Vh) rGel::Fd′ + AB1123 2,606 2,777 2,167 4 K K::RMA::rGel +AB1140 1,545 1,545 417 2 Fd rGel 1056 13,350 40,233 43,048 6 8B2.3Fab-m-1057 12,400 13,174 14,339 11 rGel_(A50(C44)) *Results represent singlevalues and not a mean value. ⁺rGel::SLT::Fd′ + k(1) and rGel::SLT::Fd′ +k(3) are separate fractions from the final purification column.

TABLE 16c PBMC HSB2 IC50, Fusion IC50, pM Plasmid Immunotoxin pM Toxin n= Toxin n = pING4644 V_(L)V_(H)::RMA::Gel 1933 4 1513 29 pING3784Gel::kappa, Fd >12,500 3 2645 7 pING3789 Gel::Fd, kappa 1212 1 3665 1pING3348 V_(L)V_(H)::Gel 2158 4 1264 9 pING3350 Gel::V_(H)V_(L) 8056 32729 4 pING3775 (Gel::RMA::kappa, Fd′)₂ 175 1 44 22 pING3770Gel::RMA::k, 3548 2 519 9 Gel::RMA::Fd pING3772 Gel::SLT::k,Gel::SLT::Fd — — 663 6

The results presented in Tables 16a, 16b and 16c demonstrate thatgelonin immunofusions may vary in their activity. In general,immunofusions of the invention which have IC₅₀ median or mean values ofless than 2000 pM Toxin display strong activity; whereas those with IC₅₀values equal to or less than 500 pM Toxin are considered highly active.In sum, the results in Tables 16a, 16b and 16c demonstrate that theoptimum fusion protein for killing a particular cell line may varydepending upon the targeted call.

EXAMPLE 22

Preparation of BRIP

BRIP possesses characteristics which make it an attractive candidate fora component of immunotoxins. BRIP is a naturally unglycosylated proteinthat may have reduced uptake in the liver and enhanced circulatoryresidence time in vivo. Additionally, BRIP is less toxic and lessimmunogenic in animals than the A-chain of ricin. Cloning of the BRIPgene and expression of recombinant BRIP in an E. coli expression systemobviates the need to purify native BRIP directly from barley, andenables the development of analogs of BRIP which may be conjugated withan available cysteine residue for conjugation to antibodies.

A. Purification of BRIP and Generation of Polyclonal Antibodies to BRIP

Native BRIP was purified from pearled barley flour. Four kilograms offlour was extracted with 16 liters of extraction buffer (10 mM NaPO4, 25mM NaCl, pH 7.2) for 20 hours at 4° C. The sediment was removed bycentrifugation, and 200 ml of packed S-Sepharose (Pharmacia, Piscataway,N.J.) was added to absorb BRIP. After mixing for 20 hours at 4° C., theresin was allowed to settle out, rinsed several times with extractionbuffer and then packed into a 2.6×40 cm column. Once packed, the columnwas washed with extraction buffer (150 ml/h) until the absorbance of theeffluent approached zero. BRIP was then eluted with a linear gradient of0.025 to 0.3 M NaCl in extraction buffer and 5 ml fractions werecollected. BRIP-containing peaks (identified by Western analysis ofcolumn fractions) were pooled, concentrated to about 20 ml, and thenchromatographed on a 2.6×100 cm Sephacryl S-200HR (Pharmacia) columnequilibrated in 10 mM NaPO₄, 125 mM NaCl, pH 7.4 (10 ml/hr).BRIP-containing peaks were pooled again, concentrated, and stored at−70° C.

The resulting purified BRIP protein had a molecular weight of about30,000 Daltons, based upon the mobility of Coomassie-stained proteinbands following SDS-PAGE. The amino acid composition was consistent withthat published by Asano et al., Carlsberg Res. Comm., 49:619-626 (1984).

Rabbits were immunized with purified BRIP to generate polyclonalantisera.

B. Cloning of the BRIP Gene

A cDNA expression library prepared from germinating barley seeds in thephage λ expression vector λZAPII was purchased from Stratagene, LaJolla, Calif. Approximately 700,000 phage plaques were screened withanti-BRIP polyclonal antisera and 6 immunoreactive plaques wereidentified. One plaque was chosen, and the CDNA contained therein wasexcised from λZAPII with EcoRI and subcloned into pUC18 generating thevector pBS1. The cDNA insert was sequenced with Sequenase (United StatesBiochemical, Cleveland, Ohio). The DNA sequence of the native BRIP geneis set out in SEQ ID NO: 12. To confirm that cDNA encoded the nativeBRIP gene, the cDNA was expressed in the E. coli plasmid pKX233-2(Pharmacia). BRIP protein was detected in IPTG-induced cells transformedwith the plasmid by Western analysis with above-described rabbitanti-BRIP antisera.

C. Construction of an E. coli Expression Vector Containing the BRIP Gene

Barley cDNA containing the BRIP gene was linked to a pelB leadersequence and placed under control of an araB promoter in a bacterialsecretion vector.

An intermediate vector containing the BRIP gene linked to the pelBleader sequence was generated. Plasmid pBS1 was cut with NcoI, treatedwith Mung Bean Nuclease, cut with BamHI and the 760 bp fragmentcorresponding to amino acids 1-256 of BRIP was purified from an agarosegel. Concurrently, a unique XhoI site was introduced downstream of the3′-end of the BRIP gene in pBS1 by PCR amplification with a pUC18 vectorprimer (identical to the Reverse® primer sold by NEB or BRL butsynthesized on a Cyclone Model 8400 DNA synthesizer) and the specificprimer BRIP 3′Xho. The sequence of each of the primers is set out below.

Reverse (SEQ ID NO: 45) 5′ AACAGCTATGACCATG 3′

BRIP 3′Xho (SEQ ID NO: 46) 5′ TGAACTCGAGGAAAACTACCTATTTCCCAC 3′

Primer BRIP 3′Xho includes a portion corresponding to the last 8 bp ofthe BRIP gene, the termination codon and several base pairs downstreamof the BRIP gene, and an additional portion that introduces a XhoI sitein the resulting PCR fragment. The PCR reaction product was digestedwith BamHI and XhoI, and an 87 bp fragment containing the 3′-end of theBRIP gene was purified on a 5% acrylamide gel. The 760 and 87 bppurified BRIP fragments were ligated in the vector pING1500 adjacent tothe pelB leader sequence. pING1500 had previously been cut with SstI,treated with T4 polymerase, cut with XhoI, and purified. The DNAsequence at the junction of the pelB leader and the 5′-end of the BRIPgene was verified by DNA sequence analysis. This vector was denotedpING3321-1.

The final expression vector was assembled by placing the BRIP gene underthe control of the inducible araB promoter. Plasmid pING3321-1 was cutwith PstI and XhoI, and the BRIP gene linked to the pelB leader waspurified from an agarose gel. The expression vector pING3217, containingthe araB promoter, was cut with PstI and XhoI and ligated to the BRIPgene. The expression vector was denoted pING3322.

Arabinose induction of E. coli cells containing the plasmid pING3322 ina fermenter resulted in the production of about 100 mg per liter ofrecombinant BRIP. E. coli-produced BRIP displays properties identical toBRIP purified directly from barley seeds.

D. Construction of BRIP Analogs with a Free Cysteine Residue

The BRIP protein contains no cysteine residues, and therefore containsno residues directly available which may form a disulfide linkage toantibodies or other proteins. Analogs of recombinant BRIP were generatedwhich contain a free cysteine residue near the C-terminus of theprotein. Three residues of the BRIP protein were targets for amino acidsubstitutions. Comparison of the amino acid sequence of BRIP to theknown tertiary structure of the ricin A-chain (see FIG. 2) suggestedthat the three positions would be available near the surface of themolecule. The three BRIP analogs include cysteines substituted in placeof serine₂₇₇, alanine₂₇₀, and leucine₂₅₆ of the native protein, and weredesignated BRIP_(C256), (SEQ ID NO: 127), BRIP_(C270) (SEQ ID NO: 128)and BRIP_(C256) (SEQ ID NO: 129), respectively.

(1) A plasmid vector capable of expressing the BRIP_(C277) analog wasconstructed by replacing the 3′-end of the BRIP gene with a DNA segmentconferring the amino acid change. The EcoRI fragment containing the BRIPgene from pBS1 was subcloned into M13mp18, and single-stranded DNA(anti-sense strand) was amplified by PCR with primers OBM2(corresponding nucleotides −11 to +8 of the BRIP gene) and OMB4(corresponding to amino acids 264-280 of BRIP and the termination codonof BRIP, and incorporating the substitution of a cysteine codon for thenative codon for serine₂₇₇ of native BRIP). The sequences of primersOBM2 and OMB4, wherein the underlined nucleotides encode the substitutedcysteine, are set out below.

OBM2 (SEQ ID NO: 47) 5′ GCATTACATCCATGGCGGC 3′

OMB4 (SEQ ID NO: 48) 5′ GATATCTCGAGTTAACTATTTCCCACCACACGCATGGAACAGCTCCAGCGCCTTGGCCACCGTC 3′

A fragment containing a BRIP gene in which the codon for the amino acidat position 277 was changed to a cysteine codon was amplified. Thefragment was cloned into the SmaI site of pUC19 (BRL) and the plasmidgenerated was denoted pMB22. pMB22 was digested with EcoRI and anEcoRI-XhoI linker (Clonetech, Palo Alto, Calif.) was ligated into thevector. Subsequent digestion with XhoI and religation generated vectorpINGMB2X. A BamHI to XhoI fragment encoding the 3′-end of BRIP with thealtered amino acid was excised from pMB2X and the fragment was purifiedon a 5% acrylamide gel. This fragment along with an EcoRI to BamHIfragment containing the pelB leader sequence and sequences encoding thefirst 256 amino acids of BRIP were substituted in a three piece ligationinto pING3322 cut with ZcoRI and XhoI. The resulting vector containingthe BRIP_(C277) analog was designated pING3803 (ATCC Accession No.68722).

(2) A BRIP analog with a free cysteine at position 256 was constructedusing PCR to introduce the amino acid substitution. A portion of theexpression plasmid pING3322 was amplified with primers BRIP-256 andHINDIII-2. The sequence of each primer is set out below.

BRIP-256 (SEQ ID NO: 49) 5′ TGTCTGTTCGTGGAGGTGCCG 3′

HINDIII-2 (SEQ ID NO: 44) 5′ CGTTAGCAATTTAACTGTGAT 3′

Nucleotides 4-21 of primer BRIP-256 encode amino acids 256-262 of BRIPwhile the underlined nucleotides specify the cysteine to be substitutedfor the leucine at the corresponding position of the native BRIPprotein. Primer HINDIII-2 corresponds to a portion of the plasmid. ThePCR product, which encodes the carboxyl terminal portion of the BRIPanalog, was treated with T4 polymerase, cut with XhoI, and the resultingfragment was purified on a 5% acrylamide gel. Concurrently, plasmidpING3322 was cut with BamHI, treated with T4 polymerase, cut with EcoRI,and the fragment containing the pelB leader sequence and sequencesencoding the first 256 amino acids of BRIP was purified. The twofragments were then assembled back into pING3322 to generate the geneencoding the analog BRIP_(C256). This plasmid is denoted pING3801.

(3) A BRIP analog with a cysteine at position 270 was also generatedusing PCR. A portion of the expression plasmid pING3322 was amplifiedwith primers BRIP-270 and the HINDIII-2 primer (SEQ ID NO: 44). Thesequence of primer BRIP-270 is set out below.

BRIP-270 (SEQ ID NO: 50) 5′ CCAAGTGTCTGGAGCTGTTCCATGCGA 3′

Primer BRIP-270 corresponds to amino acids 268-276 of BRIP with theexception of residue 270. The codon of the primer corresponding toposition 270 specifies a cysteine instead of the alanine present in thecorresponding position in native BRIP. The PCR product was treated withT4 polymerase, cut with XhoI, and the 51 bp fragment, which encodes thecarboxyl terminal portion of the analog, was purified on a 5% acrylamidegel. The fragment (corresponding to amino acids 268-276 of BRIP_(C270))was cloned in a three piece ligation along with the internal 151 bp BRIPrestriction fragment from SstII to MscI (corresponding to BRIP aminoacids 217-267) from plasmid pING3322, and restriction fragment fromSstII to XhoI from pING3322 containing the remainder of the BRIP gene.The plasmid generated contains the gene encoding the BRIPC270 analog andis designated pING3802.

E. Purification of Recombinant BRIP and the BRIP Analogs

Recombinant BRIP (rBRIP) and the BRIP analogs with free cysteineresidues were purified essentially as described for native BRIP exceptthey were prepared from concentrated fermentation broths. For rBRIP,concentrated broth from a 10 liter fermentation batch was exchanged into10 mM Tris, 20 mM NaCl pH 7.5, loaded onto a Sephacryl S-200 column, andeluted with a 20 to 500 mM NaCl linear gradient. Pooled rBRIP wasfurther purified on a Blue Toyopearl® column (TosoHaas) loaded in 20 mMNaCl and eluted in a 20 to 500 mM NaCl gradient in 10 mM Tris, pH 7.5.For BRIP analogs, concentrated fermentation broths were loaded onto aCM52 column (Whatman) in 10 mM phosphate buffer, pH 7.5, and eluted witha 0 to 0.3M NaCl linear gradient. Further purification was bychromatography on a Blue Toyopearl® column.

F. Reticulocyte Lysate Assay

The ability of the rBRIP and the BRIP analogs to inhibit proteinsynthesis in vitro was tested by reticulocyte lysate assay as describedin Example 1. Serial log dilutions of standard toxin (RTA 30), nativeBRIP, rBRIP and BRIP analogs were tested over a range of 1 μg/ml to 1pg/ml. By comparison with an uninhibited sample, the picomolarconcentration of toxin (pM) which corresponds to 50% inhibition ofprotein synthesis (IC₅₀) was calculated. The results of the assays arepresented below in Table 17.

TABLE 17 Toxin IC₅₀ (pM) RTA 30 3.1 Native BRIP 15 rBRIP 18 BRIP_(C256)23 BRIP_(C270) 20 BRIP_(C277) 24

The RLA results indicate that the BRIP analogs exhibitribosome-inactivating activity comparable to that of the recombinant andnative BRIP toxin. All the analogs retained the natural ability ofnative BRIP to inhibit protein synthesis, suggesting that amino acidsubstitution at these positions does not affect protein folding andactivity.

EXAMPLE 23

Construction of BRIP Immunoconjugates

Immunoconjugates of native BRIP (SEQ ID NO: 3) with 4A2 (described inMorishima at al., J. Immunol., 129:1091 (1982) and H65 antibody(obtained from hybridoma ATCC HB9286) which recognize the T-celldeterminants CD7 and CD5, respectively, were constructed.Immunoconjugates of ricin A-chains (RTAs) with 4A2 and H65 antibody wereconstructed as controls. The H65 antibody and ricin A-chains as well asthe RTA immunoconjugates were prepared and purified according to methodsdescribed in U.S. patant application Ser. No. 07/306,433 supra and inInternational Publication No. WO 89/06968.

To prepare immunoconjugates of native BRIP, both the antibody (4A2 orH65) and native BRIP were chemically modified with the hindered linker5-methyl-2-iminothiolane (M2IT) at lysine residues to introduce areactive sulfhydryl group as described in Goff et al., BioconjugateChem., 1:381-386 (1990). BRIP (3 mg/ml) was first incubated with 0.5 mMM2IT and 1 mM DTNB in 25 mM triethanolamine, 150 mM NaCl, pH 8.0, for 3hours at 25° C. The derivitized BRIP-(M2IT)-S-S-TNB was then desalted ona column of Sephadex GF-05LS and the number of thiol groups introducedwas quantitated by the addition of 0.1 mM DTT. On average, each BRIPmolecule contained 0.7 SH/mol.

4A2 or H65 antibody (4 mg/ml) in triethanolamine buffer was similarlyincubated with M2IT (0.3 mM) and DTNB (1 mM) for 3 hours at 25° C.Antibody-(M2IT)-S-S-TNB was then desalted and the TNB:antibody ratio wasdetermined. To prepare the conjugate, the BRIP-(M2IT)-S-S-TNB was firstreduced to BRIP—(M2IT)-SH by treatment with 0.5 mM DTT for 1 hour at 25°C., desalted by gel filtration of Sephadex® GF-05LS to remove thereducing agent, and then mixed with antibody-(M2IT)-S-S-TNB.

Following a 3 hour incubation at 25° C., and an additional 18 hours at4° C., the conjugate was purified by sequential chromatography on AcA44(IBF) and Blue Toyopearl®. Samples of the final product were run on 5%non-reducing SDS PAGE, Coomassie stained, and scanned with a Shimadzulaser densitometer to quantitate the number of toxins per antibody.

The BRIP analogs containing a free cysteine were also conjugated to 4A2and H65 antibodies. The analogs were treated with 50 mM DTT either for 2hours at 25° C. or for 18 hours at 4° C. to expose the reactivesulfhydryl group of the cysteine and desalted. The presence of a freesulfhydryl was verified by reaction with DTNB [Ellman et al., Arch.Biochem. Biophys, 82:70-77 (1959)]. 4A2 or H65 antibody derivatized asdescribed above with M2IT was incubated with the reduced BRIP analogs ata ratio of 1:5 at room temperature for 3 hours and then overnight at 4°C. Immunoconjugates H65-BRIP_(C256), 4A2-BRIP_(C256), H65-BRIP_(C277)were prepared in 25 mM triethanolamine, 150 mM NaCl pH 8, whileimmunoconjugates H65-BRIP_(C270), 4A2-BRIP_(C270) and 4A2-BRIP_(C277)were prepared in 0.1 M sodium phosphate, 150 mM NaCl pH 7.5. Followingconjugation, 10 μM mercaptoethylamine was added for 15 minutes at 25° C.to quenched any unreacted m2IT linkers on the antibody. The quenchedreaction solution was promptly loaded onto a gel filtration column(AcA44) to remove unconjugated ribosome-inactivating protein.Purification was completed using soft gel affinity chromatography onBlue Toyopearl® resin using a method similar to Knowles et al., Analyt.Biochem., 160:440 (1987). Samples of the final product were run on 5%non-reduced SDS PAGE, Coomassie stained, and scanned with a Shimadzulaser densitometer to quantitate the number of toxins per antibody. Theconjugation efficiency was substantially greater for BRIP_(C277) (78%)than for either of the other two analogs, BRIP_(C270) and BRIP_(C256)(each of these was about 10%). Additionally, the BRIP_(C277) product wasa polyconjugate, i.e., several BRIP molecules conjugated to a singleantibody, in contrast to the BRIP_(C270) and BRIP_(C256) products whichwere monoconjugates.

EXAMPLE 24

Properties of BRIP Immunoconjugates

A. Whole Cell Kill Assay

Immunoconjugates of native BRIP and of the BRIP analogs were tested forthe ability to inhibit protein synthesis in HSB2 cells by the whole cellkill assay described in Example 1. Standard immunoconjugates H65-RTA(H65 derivatized with SPDP linked to RTA) and 4MRTA (4A2 antibodyderivatized with M2IT linked to RTA) and BRIP immunoconjugate sampleswere diluted with RPMI without leucine at half-log concentrationsranging from 2000 to 0.632 ng/ml. All dilutions were added in triplicateto microtiter plates containing 1×10⁵ HSB2 cells. HSB2 plates wereincubated for 20 hours at 37° C. and then pulsed with ³H-Leu for 4 hoursbefore harvesting. Samples were counted on the Inotec Trace 96 cascadeionization counter. By comparison with an untreated sample, thepicomolar toxin concentration (pM T) of immunoconjugate which resultedin a 50% inhibition of protein synthesis (IC₅₀) was calculated. Theassay results are presented below in Table 18.

TABLE 18 Conjugate IC₅₀ (pM T) 4A2-BRIP 122.45 4A2-BRIP_(C270) 46.34A2-BRIP_(C277) 57.5 4A2-BRIP_(C256) 1116 H65-BRIP >5000 H65-BRIP_(C277)1176

The BRIP analog conjugates were less potent than the ricin conjugatecontrol (data not shown). The immunotoxins containing antibody 4A2 andeither the BRIP_(C270) or the BRIP_(C277) analog exhibited comparable toincreased specific cytotoxicity toward target cells as compared toimmunotoxin containing native BRIP. While 4A2-BRIP_(C256) is less activethan 4A2-BRIP, 4A2-BRIP_(C270) and 4A2-BRIP_(C277) are between 3 and 4times more active. Similarly, the immunoconjugate of H65 to BRIP_(C277)shows greater toxicity toward target cells than the immunoconjugate ofH65 to native BRIP. Thus, linkage of antibody to BRIP derivatives whichhave an available cysteine residue in an appropriate location results inimmunotoxins with enhanced specific toxicity toward target cellsrelative to conjugates with native BRIP.

B. Disulfide Bond Stability Assay

Immunoconjugates prepared with native BRIP and the BRIP analogs wereexamined by the disulfide bond stability assay described in Example 1.Briefly, conjugates were incubated with increasing concentrations ofglutathione for 1 hour at 37° C. and, after terminating the reactionwith iodoacetamide, the amount of RIP released was quantitated bysize-exclusion HPLC on a TosoHaas TSK-G2000SW column.

By comparisons with the amount of RIP released by high concentrations of2-mercaptoethanol (to determine 100% release), the concentration ofglutathione required to release 50% of the RIP (the RC₅₀) wascalculated. As shown below in Table 19, the conjugates prepared withBRIP_(C270) or BRIP_(C277) were significantly more stable than eitherthe RTA conjugates or those prepared with native BRIP.

TABLE 19 Conjugate RC₅₀ (mM) H65-RTA 7.0 H65-BRIP 2.8 H65-BRIPC277 196.04A2-RTA 4.4 4A2-BRIP 3.3 42-BRIP_(C270) 53.0 4A2-BRIP_(C277) 187.0

These unexpected results suggest that conjugates prepared with Type IRIP analogs according to the present invention may have enhancedstability and efficacy in vivo.

EXAMPLE 25

Preparation of Momordin and Analogs Thereof

Plants of the genus Momordica produce a number of related proteins knownas momordins or momorcharins which are Type I RIPs. The gene encodingmomordin II was cloned from Momordica balsamina seeds.

A. Preparation of M. balsamina RNA

Total RNA was prepared from 4 g of M. balsamina seeds as described inAusubel et al., supra. PolyA containing RNA was prepared from 1 mg oftotal RNA by chromatography on oligo-(dT)-cellulose. 40 mg ofoligo-(dT)-cellulose Type 7 (Pharmacia) was added to 0.1 N NaOH andpoured into a disposable column (Biorad). The column was washed withwater until the eluate was pH 5.5, and then was washed with 1×loadingbuffer (50 mM NaCitrate, 0.5 M NaCl, 1 mM EDTA, 0.1% SDS, pH 7.0) untilthe eluate was pH 7.0. 1 mg of total RNA was suspended in 300 μl ofwater, heated to 65° C. for 5 minutes, and 300 μl of 2×loading bufferwas added (100 mM Na Citrate, 1 M NaCl, 2 mM EDTA, and 0. 2% SDS) TheRNA was loaded onto the column, and the flow through was reheated to 65°C., cooled to room temperature, and reloaded onto the column.Column-bound mRNA was washed 5 times with 0.5 ml of 1×loading buffer,and two times with 0.5 ml of 0.05M NaCitrate, 0.1 M NaCl, 1 mM EDTA,0.1% SDS. PolyA-containing RNA was eluted two times from the column with0.5 ml of 25 mM NaCitrate, 1 mM EDTA, and 0.05% SDS.

B. Library Preparation

A cDNA library from the polyA-containing M. balsamina RNA was preparedin a bacterial expression plasmid with the SuperScript Plasmid System(BRL, Gaithersburg, Md.). The cDNA was synthesized from 2 μg of polyA-containing RNA, size fractionated, digested with NotI, and ligatedinto the expression vector pSPORT as recommended by the manufacturer ofthe vector, BRL.

C. Cloning of the Momordin II Gene

A DNA fragment encoding the first 27 amino acids of momordin II wasamplified from M. balsamina cDNA by PCR. First strand cDNA was preparedfrom 100 ng of polyA containing RNA with an RNA-PCR Kit (Perkin ElmerCetus). Two partially degenerate primers were synthesized based on theamino acid sequence of the first 27 amino acids of momordin II describedin Li et al., Experientia, 36:524-527 (1980). Because the amino acidsequence of amino acids 1-27 of momordin II is 52% homologous to aminoacids 1-17 of momordin I [Ho et al., BBA, 1088:311-314 (1991)], somecodon assignments in the degenerate primers were based on homology tothe corresponding amino acid as well as codon preference in the momordinI gene. The sequences of primers momo-3 and momo-4 are set out belowusing IUPAC nucleotide symbols.

momo-3 (SEQ ID NO: 51) 5′ GATGTTAAYTTYGAYTTGTCNACDGCTAC 3′

momo-4 (SEQ ID NO: 52) 5′ ATTGGNAGDGTAGCCCTRAARTCYTCDAT 3′

The resulting 81 bp PCR product was purified on a 5% acrylamide gel andcloned into the Smal site of pUC18. Three candidate clones weresequenced, and one clone, pMO110, was identified which encoded theN-terminal 27 amino acids of momordin II.

A hybridization probe was designed for screening of the momordin II cDNAlibrary based on the sequence of the pMO100 momordin II DNA fragment.The sequence of the primer momo-5 is shown below.

momo-5 (SEQ ID NO: 53) 5′ GCCACTGCAAAAACCTACACAAAATTTATTGA 3′

Primer momo-5 corresponds to amino acids 9-18 of mature momordin II. Theunderlined nucleotides of the primer were expected to match the DNAsequence of the momordin II gene exactly. Since this sequence is highlyA/T-rich and may hybridize to the momordin II gene weakly, theadditional adjacent nucleotides were included in the primer. Bases 3 and30 (overlined) were in the “wobble” position (i.e., the third nucleotidein a codon) of amino acids 9 (alanine) and 18 (isoleucine),respectively, of momordin II and may not be identical to the nucleotidebases in the native gene.

A 90,000 member cDNA library in pSPORT was screened with ³²P-kinasedmomo-5, and eight potential candidate clones were identified. One clone,pING3619, was sequenced and contains an open reading frame correspondingin part to the expected N-terminal 27 residues of Momordin II. Thecomplete momordin gene contains 286 amino acids, the first 23 of whichare a presumed leader signal (mature momordin II is 263 residues). TheDNA sequence of the momordin II gene is set out in SEQ ID NO: 13.

D. Construction of an Expression Vector Containing the Momordin II Gene

A bacterial expression vector for the momordin II gene was constructed.Two PCR primers were synthesized, one (momo-9) which primes from the +1residue of the mature momordin II amino acid sequence, and one at theC-terminus (momo-10) of momordin II which introduces an XhoI restrictionsite:

momo-9 (SEQ ID NO: 54) 5′ GATGTTAACTTCGATTTGTCGA 3′

momo-10 (SEQ ID NO: 55) 5′ TCAACTCGAGGTACTCAATTCACAACAGATTCC 3′

pING3619 was amplified with momo-9 and momo-10, and the product wastreated with T4 polymerase, cut with XhoI, and purified on an agarosegel. This gene fragment was ligated along with the 131 bp pelB leaderfragment from pIC100 which has been generated by SstI digestion,T4-polymerase treatment, and EcoRI digestion, into the araB expressionvector cleaved with EcoRI and XhoI. The product of this three pieceligation was sequenced to verify that the pelB junction and momordin IIcoding sequence were correct. Arabinose induction of calls containingthe momordin II expression plasmid pING3621 results in production ofmomordin II in E. coli.

E. Analogs of Mormordin II

Mormordin II has no natural cysteines available for conjugation toantibody. Analogs of momordin which have a free cysteine for conjugationto an antibody may be constructed. Positions likely to be appropriatefor substitution of a cysteine residue may be identified from FIG. 3 aspositions near the ricin A-chain cysteine₂₅₉ and as positions includingthe last 26 amino acids of momordin II that are accessible to solvent.For example, the arginine at position 242 of momordin II aligns with thericin A-chain cysteine at position 259 and is a preferred target forsubstitution. Additional preferred substitution positions for momordinII include the serine at position 241 and the alanine at position 243.

While the present invention has been described in terms of preferredembodiments, it is understood that variations and improvements willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations which come withinthe scope of the invention as claimed.

173 267 amino acids amino acid linear protein 1 Ile Phe Pro Lys Gln TyrPro Ile Ile Asn Phe Thr Thr Ala Gly Ala 1 5 10 15 Thr Val Gln Ser TyrThr Asn Phe Ile Arg Ala Val Arg Gly Arg Leu 20 25 30 Thr Thr Gly Ala AspVal Arg His Glu Ile Pro Val Leu Pro Asn Arg 35 40 45 Val Gly Leu Pro IleAsn Gln Arg Phe Ile Leu Val Glu Leu Ser Asn 50 55 60 His Ala Glu Leu SerVal Thr Leu Ala Leu Asp Val Thr Asn Ala Tyr 65 70 75 80 Val Val Gly TyrArg Ala Gly Asn Ser Ala Tyr Phe Phe His Pro Asp 85 90 95 Asn Gln Glu AspAla Glu Ala Ile Thr His Leu Phe Thr Asp Val Gln 100 105 110 Asn Arg TyrThr Phe Ala Phe Gly Gly Asn Tyr Asp Arg Leu Glu Gln 115 120 125 Leu AlaGly Asn Leu Arg Glu Asn Ile Glu Leu Gly Asn Gly Pro Leu 130 135 140 GluGlu Ala Ile Ser Ala Leu Tyr Tyr Tyr Ser Thr Gly Gly Thr Gln 145 150 155160 Leu Pro Thr Leu Ala Arg Ser Phe Ile Ile Cys Ile Gln Met Ile Ser 165170 175 Glu Ala Ala Arg Phe Gln Tyr Ile Glu Gly Glu Met Arg Thr Arg Ile180 185 190 Arg Tyr Asn Arg Arg Ser Ala Pro Asp Pro Ser Val Ile Thr LeuGlu 195 200 205 Asn Ser Trp Gly Arg Leu Ser Thr Ala Ile Gln Glu Ser AsnGln Gly 210 215 220 Ala Phe Ala Ser Pro Ile Gln Leu Gln Arg Arg Asn GlySer Lys Phe 225 230 235 240 Ser Val Tyr Asp Val Ser Ile Leu Ile Pro IleIle Ala Leu Met Val 245 250 255 Tyr Arg Cys Ala Pro Pro Pro Ser Ser GlnPhe 260 265 251 amino acids amino acid linear protein 2 Gly Leu Asp ThrVal Ser Phe Ser Thr Lys Gly Ala Thr Tyr Ile Thr 1 5 10 15 Tyr Val AsnPhe Leu Asn Glu Leu Arg Val Lys Leu Lys Pro Glu Gly 20 25 30 Asn Ser HisGly Ile Pro Leu Leu Arg Lys Lys Cys Asp Asp Pro Gly 35 40 45 Lys Cys PheVal Leu Val Ala Leu Ser Asn Asp Asn Gly Gln Leu Ala 50 55 60 Glu Ile AlaIle Asp Val Thr Ser Val Tyr Val Val Gly Tyr Gln Val 65 70 75 80 Arg AsnArg Ser Tyr Phe Phe Lys Asp Ala Pro Asp Ala Ala Tyr Glu 85 90 95 Gly LeuPhe Lys Asn Thr Ile Lys Thr Arg Leu His Phe Gly Gly Ser 100 105 110 TyrPro Ser Leu Glu Gly Glu Lys Ala Tyr Arg Glu Thr Thr Asp Leu 115 120 125Gly Ile Glu Pro Leu Arg Ile Gly Ile Lys Lys Leu Asp Glu Asn Ala 130 135140 Ile Asp Asn Tyr Lys Pro Thr Glu Ile Ala Ser Ser Leu Leu Val Val 145150 155 160 Ile Gln Met Val Ser Glu Ala Ala Arg Phe Thr Phe Ile Glu AsnGln 165 170 175 Ile Arg Asn Asn Phe Gln Gln Arg Ile Arg Pro Ala Asn AsnThr Ile 180 185 190 Ser Leu Glu Asn Lys Trp Gly Lys Leu Ser Phe Gln IleArg Thr Ser 195 200 205 Gly Ala Asn Gly Met Phe Ser Glu Ala Val Glu LeuGlu Arg Ala Asn 210 215 220 Gly Lys Lys Tyr Tyr Val Thr Ala Val Asp GlnVal Lys Pro Lys Ile 225 230 235 240 Ala Leu Leu Lys Phe Val Asp Lys AspPro Lys 245 250 280 amino acids amino acid linear protein 3 Ala Ala LysMet Ala Lys Asn Val Asp Lys Pro Leu Phe Thr Ala Thr 1 5 10 15 Phe AsnVal Gln Ala Ser Ser Ala Asp Tyr Ala Thr Phe Ile Ala Gly 20 25 30 Ile ArgAsn Lys Leu Arg Asn Pro Ala His Phe Ser His Asn Arg Pro 35 40 45 Val LeuPro Pro Val Glu Pro Asn Val Pro Pro Ser Arg Trp Phe His 50 55 60 Val ValLeu Lys Ala Ser Pro Thr Ser Ala Gly Leu Thr Leu Ala Ile 65 70 75 80 ArgAla Asp Asn Ile Tyr Leu Glu Gly Phe Lys Ser Ser Asp Gly Thr 85 90 95 TrpTrp Glu Leu Thr Pro Gly Leu Ile Pro Gly Ala Thr Tyr Val Gly 100 105 110Phe Gly Gly Thr Tyr Arg Asp Leu Leu Gly Asp Thr Asp Lys Leu Thr 115 120125 Asn Val Ala Leu Gly Arg Gln Gln Leu Ala Asp Ala Val Thr Ala Leu 130135 140 His Gly Arg Thr Lys Ala Asp Lys Ala Ser Gly Pro Lys Gln Gln Gln145 150 155 160 Ala Arg Glu Ala Val Thr Thr Leu Val Leu Met Val Asn GluAla Thr 165 170 175 Arg Phe Gln Thr Val Ser Gly Phe Val Ala Gly Leu LeuHis Pro Lys 180 185 190 Ala Val Glu Lys Lys Ser Gly Lys Ile Gly Asn GluMet Lys Ala Gln 195 200 205 Val Asn Gly Trp Gln Asp Leu Ser Ala Ala LeuLeu Lys Thr Asp Val 210 215 220 Lys Pro Pro Pro Gly Lys Ser Pro Ala LysPhe Ala Pro Ile Glu Lys 225 230 235 240 Met Gly Val Arg Thr Ala Glu GlnAla Ala Asn Thr Leu Gly Ile Leu 245 250 255 Leu Phe Val Glu Val Pro GlyGly Leu Thr Val Ala Lys Ala Leu Glu 260 265 270 Leu Phe His Ala Ser GlyGly Lys 275 280 263 amino acids amino acid linear protein 4 Asp Val AsnPhe Asp Leu Ser Thr Ala Thr Ala Lys Thr Tyr Thr Lys 1 5 10 15 Phe IleGlu Asp Phe Arg Ala Thr Leu Pro Phe Ser His Lys Val Tyr 20 25 30 Asp IlePro Leu Leu Tyr Ser Thr Ile Ser Asp Ser Arg Arg Phe Ile 35 40 45 Leu LeuAsp Leu Thr Ser Tyr Ala Tyr Glu Thr Ile Ser Val Ala Ile 50 55 60 Asp ValThr Asn Val Tyr Val Val Ala Tyr Arg Thr Arg Asp Val Ser 65 70 75 80 TyrPhe Phe Lys Glu Ser Pro Pro Glu Ala Tyr Asn Ile Leu Phe Lys 85 90 95 GlyThr Arg Lys Ile Thr Leu Pro Tyr Thr Gly Asn Tyr Glu Asn Leu 100 105 110Gln Thr Ala Ala His Lys Ile Arg Glu Asn Ile Asp Leu Gly Leu Pro 115 120125 Ala Leu Ser Ser Ala Ile Thr Thr Leu Phe Tyr Tyr Asn Ala Gln Ser 130135 140 Ala Pro Ser Ala Leu Leu Val Leu Ile Gln Thr Thr Ala Glu Ala Ala145 150 155 160 Arg Phe Lys Tyr Ile Glu Arg His Val Ala Lys Tyr Val AlaThr Asn 165 170 175 Phe Lys Pro Asn Leu Ala Ile Ile Ser Leu Glu Asn GlnTrp Ser Ala 180 185 190 Leu Ser Lys Gln Ile Phe Leu Ala Gln Asn Gln GlyGly Lys Phe Arg 195 200 205 Asn Pro Val Asp Leu Ile Lys Pro Thr Gly GluArg Phe Gln Val Thr 210 215 220 Asn Val Asp Ser Asp Val Val Lys Gly AsnIle Lys Leu Leu Leu Asn 225 230 235 240 Ser Arg Ala Ser Thr Ala Asp GluAsn Phe Ile Thr Thr Met Thr Leu 245 250 255 Leu Gly Glu Ser Val Val Asn260 248 amino acids amino acid linear protein 5 Asp Val Arg Phe Ser LeuSer Gly Ser Ser Ser Thr Ser Tyr Ser Lys 1 5 10 15 Phe Ile Gly Asp LeuArg Lys Ala Leu Pro Ser Asn Gly Thr Val Tyr 20 25 30 Asn Leu Thr Ile LeuLeu Ser Ser Ala Ser Gly Ala Ser Arg Tyr Thr 35 40 45 Leu Met Thr Leu SerAsn Tyr Asp Gly Lys Ala Ile Thr Val Ala Val 50 55 60 Asp Val Ser Gln LeuTyr Ile Met Gly Tyr Leu Val Asn Ser Thr Ser 65 70 75 80 Tyr Phe Phe AsnGlu Ser Asp Ala Lys Leu Ala Ser Gln Tyr Val Phe 85 90 95 Lys Gly Ser ThrIle Val Thr Leu Pro Tyr Ser Gly Asn Tyr Glu Lys 100 105 110 Leu Gln ThrAla Ala Gly Lys Ile Arg Glu Lys Ile Pro Leu Gly Phe 115 120 125 Pro AlaLeu Asp Ser Ala Leu Thr Thr Ile Phe His Tyr Asp Ser Thr 130 135 140 AlaAla Ala Ala Ala Phe Leu Val Ile Leu Gln Thr Thr Ala Glu Ala 145 150 155160 Ser Arg Phe Lys Tyr Ile Glu Gly Gln Ile Ile Glu Arg Ile Ser Lys 165170 175 Asn Gln Val Pro Ser Leu Ala Thr Ile Ser Leu Glu Asn Ser Leu Trp180 185 190 Ser Ala Leu Ser Lys Gln Ile Gln Leu Ala Gln Thr Asn Asn GlyThr 195 200 205 Phe Lys Thr Pro Val Val Ile Thr Asp Asp Lys Gln Gln ArgVal Glu 210 215 220 Ile Thr Asn Val Thr Ser Lys Val Val Thr Lys Asn IleGln Leu Leu 225 230 235 240 Leu Asn Tyr Lys Gln Asn Val Ala 245 247amino acids amino acid linear protein 6 Asp Val Ser Phe Arg Leu Ser GlyAla Thr Ser Ser Ser Tyr Gly Val 1 5 10 15 Phe Ile Ser Asn Leu Arg LysAla Leu Pro Asn Glu Arg Lys Leu Tyr 20 25 30 Asp Ile Pro Leu Leu Arg SerSer Leu Pro Gly Ser Gln Arg Tyr Ala 35 40 45 Leu Ile His Leu Thr Asn TyrAla Asp Glu Thr Ile Ser Val Ala Ile 50 55 60 Asp Val Thr Asn Val Tyr IleMet Gly Tyr Arg Ala Gly Asp Thr Ser 65 70 75 80 Tyr Phe Phe Asn Glu AlaSer Ala Thr Glu Ala Ala Lys Tyr Val Phe 85 90 95 Lys Asp Ala Met Arg LysVal Thr Leu Pro Tyr Ser Gly Asn Tyr Glu 100 105 110 Arg Leu Gln Thr AlaAla Gly Lys Ile Arg Glu Asn Ile Pro Leu Gly 115 120 125 Leu Pro Ala LeuAsp Ser Ala Ile Thr Thr Leu Phe Tyr Tyr Asn Ala 130 135 140 Asn Ser AlaAla Ser Ala Leu Met Val Leu Ile Gln Ser Thr Ser Glu 145 150 155 160 AlaAla Arg Tyr Lys Phe Ile Glu Gln Gln Ile Gly Lys Arg Val Asp 165 170 175Lys Thr Phe Leu Pro Ser Leu Ala Ile Ile Ser Leu Glu Asn Ser Trp 180 185190 Ser Ala Leu Ser Lys Gln Ile Gln Ile Ala Ser Thr Asn Asn Gly Gln 195200 205 Phe Glu Ser Pro Val Val Leu Ile Asn Ala Gln Asn Gln Val Ala Thr210 215 220 Ile Thr Asn Val Asp Ala Gly Val Val Thr Ser Asn Ile Ala LeuLeu 225 230 235 240 Leu Asn Arg Asn Asn Met Ala 245 263 amino acidsamino acid linear protein 7 Asp Val Ser Phe Arg Leu Ser Gly Ala Asp ProArg Ser Tyr Gly Met 1 5 10 15 Phe Ile Lys Asp Leu Arg Asn Ala Leu ProPhe Arg Glu Lys Val Tyr 20 25 30 Asn Ile Pro Leu Leu Leu Pro Ser Val SerGly Ala Gly Arg Tyr Leu 35 40 45 Leu Met His Leu Phe Asn Tyr Asp Gly LysThr Ile Thr Val Ala Val 50 55 60 Asp Val Thr Asn Val Tyr Ile Met Gly TyrLeu Ala Asp Thr Thr Ser 65 70 75 80 Tyr Phe Phe Asn Glu Pro Ala Ala GluLeu Ala Ser Gln Tyr Val Phe 85 90 95 Arg Asp Ala Arg Arg Lys Ile Thr LeuPro Tyr Ser Gly Asn Tyr Glu 100 105 110 Arg Leu Gln Ile Ala Ala Gly LysPro Arg Glu Lys Ile Pro Ile Gly 115 120 125 Leu Pro Ala Leu Asp Ser AlaIle Ser Thr Leu Leu His Tyr Asp Ser 130 135 140 Thr Ala Ala Ala Gly AlaLeu Leu Val Leu Ile Gln Thr Thr Ala Glu 145 150 155 160 Ala Ala Arg PheLys Tyr Ile Glu Gln Gln Ile Gln Glu Arg Ala Tyr 165 170 175 Arg Asp GluVal Pro Ser Leu Ala Thr Ile Ser Leu Glu Asn Ser Trp 180 185 190 Ser GlyLeu Ser Lys Gln Ile Gln Leu Ala Gln Gly Asn Asn Gly Ile 195 200 205 PheArg Thr Pro Ile Val Leu Val Asp Asn Lys Gly Asn Arg Val Gln 210 215 220Ile Thr Asn Val Thr Ser Lys Val Val Thr Ser Asn Ile Gln Leu Leu 225 230235 240 Leu Asn Thr Arg Asn Ile Ala Glu Gly Asp Asn Gly Asp Val Ser Thr245 250 255 Thr His Gly Phe Ser Ser Thr 260 250 amino acids amino acidlinear protein 8 Ala Pro Thr Leu Glu Thr Ile Ala Ser Leu Asp Leu Asn AsnPro Thr 1 5 10 15 Thr Tyr Leu Ser Phe Ile Thr Asn Ile Arg Thr Lys ValAla Asp Lys 20 25 30 Thr Glu Gln Cys Thr Ile Gln Lys Ile Ser Lys Thr PheThr Gln Arg 35 40 45 Tyr Ser Tyr Ile Asp Leu Ile Val Ser Ser Thr Gln LysIle Thr Leu 50 55 60 Ala Ile Asp Met Ala Asp Leu Tyr Val Leu Gly Tyr SerAsp Ile Ala 65 70 75 80 Asn Asn Lys Gly Arg Ala Phe Phe Phe Lys Asp ValThr Glu Ala Val 85 90 95 Ala Asn Asn Phe Phe Pro Gly Ala Thr Gly Thr AsnArg Ile Lys Leu 100 105 110 Thr Phe Thr Gly Ser Tyr Gly Asp Leu Glu LysAsn Gly Gly Leu Arg 115 120 125 Lys Asp Asn Pro Leu Gly Ile Phe Arg LeuGlu Asn Ser Ile Val Asn 130 135 140 Ile Tyr Gly Lys Ala Gly Asp Val LysLys Gln Ala Lys Phe Phe Leu 145 150 155 160 Leu Ala Ile Gln Met Val SerGlu Ala Ala Arg Phe Lys Tyr Ile Ser 165 170 175 Asp Lys Ile Pro Ser GluLys Tyr Glu Glu Val Thr Val Asp Glu Tyr 180 185 190 Met Thr Ala Leu GluAsn Asn Trp Ala Lys Leu Ser Thr Ala Val Tyr 195 200 205 Asn Ser Lys ProSer Thr Thr Thr Ala Thr Lys Cys Gln Leu Ala Thr 210 215 220 Ser Pro ValThr Ile Ser Pro Trp Ile Phe Lys Thr Val Glu Glu Ile 225 230 235 240 LysLeu Val Met Gly Leu Leu Lys Ser Ser 245 250 261 amino acids amino acidlinear protein 9 Ile Asn Thr Ile Thr Phe Asp Ala Gly Asn Ala Thr Ile AsnLys Tyr 1 5 10 15 Ala Thr Phe Met Glu Ser Leu Arg Asn Glu Ala Lys AspPro Ser Leu 20 25 30 Lys Cys Tyr Gly Ile Pro Met Leu Pro Asn Thr Asn SerThr Ile Lys 35 40 45 Tyr Leu Leu Val Lys Leu Gln Gly Ala Ser Leu Lys ThrIle Thr Leu 50 55 60 Met Leu Arg Arg Asn Asn Leu Tyr Val Met Gly Tyr SerAsp Pro Tyr 65 70 75 80 Asp Asn Lys Cys Arg Tyr His Ile Phe Asn Asp IleLys Gly Thr Glu 85 90 95 Tyr Ser Asp Val Glu Asn Thr Leu Cys Pro Ser SerAsn Pro Arg Val 100 105 110 Ala Lys Pro Ile Asn Tyr Asn Gly Leu Tyr ProThr Leu Glu Lys Lys 115 120 125 Ala Gly Val Thr Ser Arg Asn Glu Val GlnLeu Gly Ile Gln Ile Leu 130 135 140 Ser Ser Asp Ile Gly Lys Ile Ser GlyGln Gly Ser Phe Thr Glu Lys 145 150 155 160 Ile Glu Ala Asp Phe Leu LeuVal Ala Ile Gln Met Val Ser Glu Ala 165 170 175 Ala Arg Phe Lys Tyr IleGlu Asn Gln Val Lys Thr Asn Phe Asn Arg 180 185 190 Asp Phe Ser Pro AsnAsp Lys Val Leu Asp Leu Glu Glu Asn Trp Gly 195 200 205 Lys Ile Ser ThrAla Ile His Asn Ser Lys Asn Gly Ala Leu Pro Lys 210 215 220 Pro Leu GluLeu Lys Asn Ala Asp Gly Thr Lys Trp Ile Val Leu Arg 225 230 235 240 ValAsp Glu Ile Lys Pro Asp Val Gly Leu Leu Asn Tyr Val Asn Gly 245 250 255Thr Cys Gln Ala Thr 260 259 amino acids amino acid linear protein 10 ValThr Ser Ile Thr Leu Asp Leu Val Asn Pro Thr Ala Gly Gln Tyr 1 5 10 15Ser Ser Phe Val Asp Lys Ile Arg Asn Asn Val Lys Asp Pro Asn Leu 20 25 30Lys Tyr Gly Gly Thr Asp Ile Ala Val Ile Gly Pro Pro Ser Lys Glu 35 40 45Lys Phe Leu Arg Ile Asn Phe Gln Ser Ser Arg Gly Thr Val Ser Leu 50 55 60Gly Leu Lys Arg Asp Asn Leu Tyr Val Val Ala Tyr Leu Ala Met Asp 65 70 7580 Asn Thr Asn Val Asn Arg Ala Tyr Tyr Phe Arg Ser Glu Ile Thr Ser 85 9095 Ala Glu Ser Thr Ala Leu Phe Pro Glu Ala Thr Thr Ala Asn Gln Lys 100105 110 Ala Leu Glu Tyr Thr Glu Asp Tyr Gln Ser Ile Glu Lys Asn Ala Gln115 120 125 Ile Thr Gln Gly Asp Gln Ser Arg Lys Glu Leu Gly Leu Gly IleAsp 130 135 140 Leu Leu Ser Thr Ser Met Glu Ala Val Asn Lys Lys Ala ArgVal Val 145 150 155 160 Lys Asp Glu Ala Arg Phe Leu Leu Ile Ala Ile GlnMet Thr Ala Glu 165 170 175 Ala Ala Arg Phe Arg Tyr Ile Gln Asn Leu ValIle Lys Asn Phe Pro 180 185 190 Asn Lys Phe Asn Ser Glu Asn Lys Val IleGln Phe Glu Val Asn Trp 195 200 205 Lys Lys Ile Ser Thr Ala Ile Tyr GlyAsp Ala Lys Asn Gly Val Phe 210 215 220 Asn Lys Asp Tyr Asp Phe Gly PheGly Lys Val Arg Gln Val Lys Asp 225 230 235 240 Leu Gln Met Gly Leu LeuMet Tyr Leu Gly Lys Pro Lys Ser Ser Asn 245 250 255 Glu Ala Asn 813 basepairs nucleic acid single linear cDNA 11 GGGCTAGATA CCGTGTCATTCTCAACCAAA GGTGCCACTT ATATTACCTA CGTGAATTTC 60 TTGAATGAGC TACGAGTTAAATTGAAACCC GAAGGTAACA GCCATGGAAT CCCATTGCTG 120 CGCAAAAAAT GTGATGATCCTGGAAAGTGT TTCGTTTTGG TAGCGCTTTC AAATGACAAT 180 GGACAGTTGG CGGAAATAGCTATAGATGTT ACAAGTGTTT ATGTGGTGGG CTATCAAGTA 240 AGAAACAGAT CTTACTTCTTTAAAGATGCT CCAGATGCTG CTTACGAAGG CCTCTTCAAA 300 AACACAATTA AAACAAGACTTCATTTTGGC GGCAGCTATC CCTCGCTGGA AGGTGAGAAG 360 GCATATAGAG AGACAACAGACTTGGGCATT GAACCATTAA GGATTGGCAT CAAGAAACTT 420 GATGAAAATG CGATAGACAATTATAAACCA ACGGAGATAG CTAGTTCTCT ATTGGTTGTT 480 ATTCAAATGG TGTCTGAAGCAGCTCGATTC ACCTTTATTG AGAACCAAAT TAGAAATAAC 540 TTTCAACAGA GAATTCGCCCGGCGAATAAT ACAATCAGCC TTGAGAATAA ATGGGGTAAA 600 CTCTCGTTCC AGATCCGGACATCAGGTGCA AATGGAATGT TTTCGGAGGC AGTTGAATTG 660 GAACGTGCAA ATGGCAAAAAATACTATGTC ACCGCAGTTG ATCAAGTAAA ACCCAAAATA 720 GCACTCTTGA AGTTCGTCGATAAAGATCCT AAAACGAGCC TTGCTGCTGA ATTGATAATC 780 CAGAACTATG AGTCATTAGTGGGCTTTGAT TAG 813 846 base pairs nucleic acid single linear cDNA 12ATGGCGGCAA AGATGGCGAA GAACGTGGAC AAGCCGCTCT TCACCGCGAC GTTCAACGTC 60CAGGCCAGCT CCGCCGACTA CGCCACCTTC ATCGCCGGCA TCCGCAACAA GCTCCGCAAC 120CCGGCGCACT TCTCCCACAA CCGCCCCGTG CTGCCGCCGG TCGAGCCCAA CGTCCCGCCG 180AGCAGGTGGT TCCACGTCGT GCTCAAGGCC TCGCCGACCA GCGCCGGGCT CACGCTGGCC 240ATCCGCGCGG ACAACATCTA CCTGGAGGGC TTCAAGAGCA GCGACGGCAC CTGGTGGGAG 300CTCACCCCGG GCCTCATCCC CGGCGCCACC TACGTCGGGT TCGGCGGCAC CTACCGCGAC 360CTCCTCGGCG ACACCGACAA GCTAACCAAC GTCGCTCTCG GCCGACAGCA GCTGGCGGAC 420GCGGTGACCG CGCTCCACGG GCGCACCAAG GCCGACAAGG CCTCCGGCCC GAAGCAGCAG 480CAGGCGAGGG AGGCGGTGAC GACGCTGGTC CTCATGGTGA ACGAGGCCAC GCGGTTCCAG 540ACGGTGTCTG GGTTCGTGGC CGGGTTGCTG CACCCCAAGG CGGTGGAGAA GAAGAGCGGG 600AAGATCGGCA ATGAGATGAA GGCCCAGGTG AACGGGTGGC AGGACCTGTC CGCGGCGCTG 660CTGAAGACGG ACGTGAAGCC TCCGCCGGGA AAGTCGCCAG CGAAGTTCGC GCCGATCGAG 720AAGATGGGCG TGAGGACGGC TGAACAGGCC GCCAACACGC TGGGGATCCT GCTGTTCGTG 780GAGGTGCCGG GTGGGTTGAC GGTGGCCAAG GCGCTGGAGC TGTTCCATGC GAGTGGTGGG 840AAATAG 846 913 base pairs nucleic acid single linear cDNA 13 CGTCCGAAAATGGTGAAATG CTTACTACTT TCTTTTTTAA TTATCGCCAT CTTCATTGGT 60 GTTCCTACTGCCAAAGGCGA TGTTAACTTC GATTTGTCGA CTGCCACTGC AAAAACCTAC 120 ACAAAATTTATCGAAGATTT CAGGGCGACT CTTCCATTTA GCCATAAAGT GTATGATATA 180 CCTCTACTGTATTCCACTAT TTCCGACTCC AGACGTTTCA TACTCCTCGA TCTTACAAGT 240 TATGCATATGAAACCATCTC GGTGGCCATA GATGTGACGA ACGTTTATGT TGTGGCGTAT 300 CGCACCCGCGATGTATCCTA CTTTTTTAAA GAATCTCCTC CTGAAGCTTA TAACATCCTA 360 TTCAAAGGTACGCGGAAAAT TACACTGCCA TATACCGGTA ATTATGAAAA TCTTCAAACT 420 GCTGCACACAAAATAAGAGA GAATATTGAT CTTGGACTCC CTGCCTTGAG TAGTGCCATT 480 ACCACATTGTTTTATTACAA TGCCCAATCT GCTCCTTCTG CATTGCTTGT ACTAATCCAG 540 ACGACTGCAGAAGCTGCAAG ATTTAAGTAT ATCGAGCGAC ACGTTGCTAA GTATGTTGCC 600 ACTAACTTTAAGCCAAATCT AGCCATCATA AGCTTGGAAA ATCAATGGTC TGCTCTCTCC 660 AACAAATCTTTTTGGCGCAG AATCAAGGAG GAAAATTTAG AAATCCTGTC GACCTTATAA 720 AACCTACCGGGGAACGGTTT CAAGTAACCA ATGTTGATTC AGATGTTGTA AAAGGTAATA 780 TCAAACTCCTGCTGAACTCC AGAGCTAGCA CTGCTGATGA AAACTTTATC ACAACCATGA 840 CTCTACTTGGGGAATCTGTT GTGAATTGAA AGTTTAATAA TCCACCCATA TCGAAATAAG 900 GCATGTTCATGAC 913 32 base pairs nucleic acid single linear DNA 14 TTYAARGAYGCNCCNGAYGC NGCNTAYGAR GG 32 32 base pairs nucleic acid single linear DNA15 ACYTGRTCNA CNGCNGTNAC RTARTAYTTY TT 32 32 base pairs nucleic acidsingle linear DNA 16 GGNYTNGAYA CNGTNWSNTT YWSNACNAAR GG 32 23 basepairs nucleic acid single linear DNA 17 AATGGTTCAA TGCCCAAGTC TGT 23 23base pairs nucleic acid single linear DNA 18 TGTCTCTCTA TATGCCTTCT CAC23 53 base pairs nucleic acid single linear DNA 19 TCAACCCGGG CTAGATACCGTGTCATTCTC AACCAAAGGT GCCACTTATA TTA 53 23 base pairs nucleic acidsingle linear DNA 20 CTTCATTTTG GCGGCACGTA TCC 23 46 base pairs nucleicacid single linear DNA 21 CTCGAGGCTG CAAGCTTACG TGGGATTTTT TTTTTTTTTTTTTTTT 46 18 base pairs nucleic acid single linear DNA 22 CTCGCTGGAAGGTGAGAA 18 25 base pairs nucleic acid single linear DNA 23 CTCGAGGCTGCAAGCTTACG TGGGA 25 35 base pairs nucleic acid single linear DNA 24TGATCTCGAG TACTATTTAG GATCTTTATC GACGA 35 22 base pairs nucleic acidsingle linear DNA 25 GTAAGCAGCA TCTGGAGCAT CT 22 21 base pairs nucleicacid single linear DNA 26 CATTCAAGAA ATTCACGTAG G 21 23 base pairsnucleic acid single linear DNA 27 GGCCTGGACA CCGTGAGCTT TAG 23 25 basepairs nucleic acid single linear DNA 28 TCGATTGCGA TCCTAAATAG TACTC 2528 base pairs nucleic acid single linear DNA 29 TTTAGGATCG CAATCGACGAACTTCAAG 28 32 base pairs nucleic acid single linear DNA 30 GTTCGTCTGTAAAGATCCTA AATAGTACTC GA 32 27 base pairs nucleic acid single linear DNA31 GGATCTTTAC AGACGAACTT CAAGAGT 27 25 base pairs nucleic acid singlelinear DNA 32 TCTTGTGCTT CGTCGATAAA GATCC 25 27 base pairs nucleic acidsingle linear DNA 33 ATCGACGAAG CACAAGAGTG CTATTTT 27 32 base pairsnucleic acid single linear DNA 34 GTAAAACCAT GCATAGCACT CTTGAAGTTC GT 3232 base pairs nucleic acid single linear DNA 35 AGTGCTATGC ATGGTTTTACTTGATCAACT GC 32 29 base pairs nucleic acid single linear DNA 36AGCACATGTG GTGCCACTTA TATTACCTA 29 33 base pairs nucleic acid singlelinear DNA 37 TAAGTGGCAC CACATGTGCT AAAGCTCACG GTG 33 25 base pairsnucleic acid single linear DNA 38 TGACTGTGGA CAGTTGGCGG AAATA 25 33 basepairs nucleic acid single linear DNA 39 GCCAACTGTC CACAGTCATT TGAAAGCGCTACC 33 36 base pairs nucleic acid single linear DNA 40 GATGATCCTGGAAAGGCTTT CGTTTTGGTA GCGCTT 36 41 base pairs nucleic acid single linearDNA 41 AAGCCTTTCC AGGATCATCA GCTTTTTTGC GCAGCAATGG G 41 23 base pairsnucleic acid single linear DNA 42 AAGCCTTTCC AGGATCATCA CAT 23 18 basepairs nucleic acid single linear DNA 43 GCGACTCTCT ACTGTTTC 18 21 basepairs nucleic acid single linear DNA 44 CGTTAGCAAT TTAACTGTGA T 21 16base pairs nucleic acid single linear DNA 45 AACAGCTATG ACCATG 16 30base pairs nucleic acid single linear DNA 46 TGAACTCGAG GAAAACTACCTATTTCCCAC 30 19 base pairs nucleic acid single linear DNA 47 GCATTACATCCATGGCGGC 19 64 base pairs nucleic acid single linear DNA 48 GATATCTCGAGTTAACTATT TCCCACCACA CGCATGGAAC AGCTCCAGCG CCTTGGCCAC 60 CGTC 64 21base pairs nucleic acid single linear DNA 49 TGTCTGTTCG TGGAGGTGCC G 2127 base pairs nucleic acid single linear DNA 50 CCAAGTGTCT GGAGCTGTTCCATGCGA 27 29 base pairs nucleic acid single linear DNA 51 GATGTTAAYTTYGAYTTGTC NACDGCTAC 29 29 base pairs nucleic acid single linear DNA 52ATTGGNAGDG TAGCCCTRAA RTCYTCDAT 29 32 base pairs nucleic acid singlelinear DNA 53 GCCACTGCAA AAACCTACAC AAAATTTATT GA 32 22 base pairsnucleic acid single linear DNA 54 GATGTTAACT TCGATTTGTC GA 22 33 basepairs nucleic acid single linear DNA 55 TCAACTCGAG GTACTCAATT CACAACAGATTCC 33 20 amino acids amino acid linear peptide 56 Cys His His His AlaSer Arg Val Ala Arg Met Ala Ser Asp Glu Phe 1 5 10 15 Pro Ser Met Cys 2020 amino acids amino acid linear peptide 57 Pro Ser Gly Gln Ala Gly AlaAla Ala Ser Glu Ser Leu Phe Ile Ser 1 5 10 15 Asn His Ala Tyr 20 22 basepairs nucleic acid single linear DNA 58 CAGCCATGGA ATCCCATTGC TG 22 28base pairs nucleic acid single linear DNA 59 CACATGTAAA ACAAGACTTCATTTTGGC 28 36 base pairs nucleic acid single linear DNA 60 TGAAGTCTTGTTTTAGATGT GTTTTTGAAG AGGCCT 36 30 base pairs nucleic acid single linearDNA 61 ATGCCATATG CAATTATAAA CCAACGGAGA 30 39 base pairs nucleic acidsingle linear DNA 62 GGTTTATAAT TGCATATGGC ATTTTCATCA AGTTTCTTG 39 33base pairs nucleic acid single linear DNA 63 CTTTCAACAA TGCATTCGCCCGGCGAATAA TAC 33 33 base pairs nucleic acid single linear DNA 64GCGAATGCAT TGTTGAAAGT TATTTCTAAT TTG 33 26 base pairs nucleic acidsingle linear DNA 65 GTTTTGTGAG GCAGTTGAAT TGGAAC 26 34 base pairsnucleic acid single linear DNA 66 TTCAACTGCC TCACAAAACA TTCCATTTGC ACCT34 24 base pairs nucleic acid single linear DNA 67 AAAAGCTGAT GATCCTGGAAAGTG 24 35 base pairs nucleic acid single linear DNA 68 TCCAGGATCATCAGCTTTTT TGCGCAGCAA TGGGA 35 321 base pairs nucleic acid single linearDNA 69 GACATCCAGA TGACTCAGTC TCCATCTTCC ATGTCTGCAT CTCTGGGAGA CAGAGTCACT60 ATCACTTGCC GGGCGAGTCA GGACATTAAT AGCTATTTAA GCTGGTTCCA GCAGAAACCA 120GGGAAATCTC CTAAGACCCT GATCTATCGT GCAAACAGAT TGGTAGATGG GGTCCCATCA 180AGGTTCAGTG GCAGTGGATC TGGGACAGAT TATACTCTCA CCATCAGCAG CCTGCAATAT 240GAAGATTTTG GAATTTATTA TTGTCAACAG TATGATGAGT CTCCGTGGAC GTTCGGTGGA 300GGCACCAAGC TTGAAATCAA A 321 354 base pairs nucleic acid single linearDNA 70 CAGATCCAGT TGGTGCAGTC TGGACCTGGC CTGAAGAAGC CTGGAGGGTC CGTCAGAATC60 TCCTGCGCAG CTTCTGGGTA TACCTTCACA AACTATGGAA TGAACTGGGT GAAGCAGGCT 120CCAGGAAAGG GTTTAAGGTG GATGGGCTGG ATAAACACCC ACACTGGAGA GCCAACATAT 180GCTGATGACT TCAAGGGACG GTTTACCTTC TCTTTGGACA CGTCTAAGAG CACTGCCTAT 240TTACAGATCA ACAGCCTCAG AGCCGAGGAC ACGGCTACAT ATTTCTGTAC AAGACGGGGT 300TACGACTGGT ACTTCGATGT CTGGGGCCAA GGGACCACGG TCACCGTCTC CTCC 354 354 basepairs nucleic acid single linear DNA 71 GAGATCCAGT TGGTGCAGTC TGGAGGAGGCCTGGTGAAGC CTGGAGGGTC CGTCAGAATC 60 TCCTGCGCAG CTTCTGGGTA TACCTTCACAAACTATGGAA TGAACTGGGT GCGCCAGGCT 120 CCAGGAAAGG GTTTAGAGTG GATGGGCTGGATAAACACCC ACACTGGAGA GCCAACATAT 180 GCTGATTCTT TCAAGGGACG GTTTACCTTCTCTTTGGACG ATTCTAAGAA CACTGCCTAT 240 TTACAGATCA ACAGCCTCAG AGCCGAGGACACGGCTGTGT ATTTCTGTAC AAGACGGGGT 300 TACGACTGGT ACTTCGATGT CTGGGGCCAAGGGACCACGG TCACCGTCTC CTCC 354 321 base pairs nucleic acid single linearDNA 72 GACATCCAGA TGACTCAGTC TCCATCTTCC CTGTCTGCAT CTGTAGGAGA CAGAGTCACT60 ATCACTTGCC GGGCGAGTCA GGACATTAAT AGCTATTTAA GCTGGTTCCA GCAGAAACCA 120GGGAAAGCTC CTAAGACCCT GATCTATCGT GCAAACAGAT TGGAATCTGG GGTCCCATCA 180AGGTTCAGTG GCAGTGGATC TGGGACAGAT TATACTCTCA CCATCAGCAG CCTGCAATAT 240GAAGATTTTG GAATTTATTA TTGTCAACAG TATGATGAGT CTCCGTGGAC GTTCGGTGGA 300GGCACCAAGC TTGAAATCAA A 321 70 base pairs nucleic acid single linear DNA73 TGTCATCATC ATGCATCGCG AGTTGCCAGA ATGGCATCTG ATGAGTTTCC TTCTATGTGC 60GCAAGTACTC 70 78 base pairs nucleic acid single linear DNA 74 TCGAGAGTACTTGCGCACAT AGAAGGAAAC TCATCAGATG CCATTCTGGC AACTCGCGAT 60 GCATGATGATGACATGCA 78 30 base pairs nucleic acid single linear DNA 75 TGTTCGGCCGCATGTCATCA TCATGCATCG 30 15 base pairs nucleic acid single linear DNA 76AGTCATGCCC CGCGC 15 18 base pairs nucleic acid single linear DNA 77TCCCGGCTGT CCTACAGT 18 37 base pairs nucleic acid single linear DNA 78TCCAGCCTGT CCAGATGGTG TGTGAGTTTT GTCACAA 37 76 base pairs nucleic acidsingle linear DNA 79 CTAACTCGAG AGTACTGTAT GCATGGTTCG AGATGAACAAAGATTCTGAG GCTGCAGCTC 60 CAGCCTGTCC AGATGG 76 20 base pairs nucleic acidsingle linear DNA 80 CTAACTCGAG AGTACTGTAT 20 36 base pairs nucleic acidsingle linear DNA 81 TCCAGCCTGT CCAGATGGAC ACTCTCCCCT GTTGAA 36 18 basepairs nucleic acid single linear DNA 82 GTACAGTGGA AGGTGGAT 18 31 basepairs nucleic acid single linear DNA 83 CATGCGGCCG ATTTAGGATC TTTATCGACGA 31 22 base pairs nucleic acid single linear DNA 84 AACATCCAGTTGGTGCAGTC TG 22 20 base pairs nucleic acid single linear DNA 85GAGGAGACGG TGACCGTGGT 20 19 base pairs nucleic acid single linear DNA 86GACATCAAGA TGACCCAGT 19 21 base pairs nucleic acid single linear DNA 87GTTTGATTTC AAGCTTGGTG C 21 31 base pairs nucleic acid single linear DNA88 ACTTCGGCCG CACCATCTGG ACAGGCTGGA G 31 723 base pairs nucleic acidsingle linear DNA 89 GACATCCAGA TGACTCAGTC TCCATCTTCC CTGTCTGCATCTGTAGGAGA CAGAGTCACT 60 ATCACTTGCC GGGCGAGTCA GGACATTAAT AGCTATTTAAGCTGGTTCCA GCAGAAACCA 120 GGGAAAGCTC CTAAGACCCT GATCTATCGT GCAAACAGATTGGAATCTGG GGTCCCATCA 180 AGGTTCAGTG GCAGTGGATC TGGGACAGAT TATACTCTCACCATCAGCAG CCTGCAATAT 240 GAAGATTTTG GAATTTATTA TTGTCAACAG TATGATGAGTCTCCGTGGAC GTTCGGTGGA 300 GGCACCAAGC TTGAGATGAA AGGTGGCGGT GGATCTGGTGGAGGTGGGTC CGGAGGTGGA 360 GGATCTGAGA TCCAGTTGGT GCAGTCTGGA GGAGGCCTGGTGAAGCCTGG AGGGTCCGTC 420 AGAATCTCCT GCGCAGCTTC TGGGTATACC TTCACAAACTATGGAATGAA CTGGGTGCGC 480 CAGGCTCCAG GAAAGGGTTT AGAGTGGATG GGCTGGATAAACACCCACAC TGGAGAGCCA 540 ACATATGCTG ATTCTTTCAA GGGACGGTTT ACCTTCTCTTTGGACGATTC TAAGAACACT 600 GCCTATTTAC AGATCAACAG CCTCAGAGCC GAGGACACGGCTGTGTATTT CTGTACAAGA 660 CGGGGTTACG ACTGGTACTT CGATGTCTGG GGCCAAGGGACCACGGTCAC CGTCTCCTCA 720 TGA 723 723 base pairs nucleic acid singlelinear DNA 90 GAGATCCAGT TGGTGCAGTC TGGAGGAGGC CTGGTGAAGC CTGGAGGGTCCGTCAGAATC 60 TCCTGCGCAG CTTCTGGGTA TACCTTCACA AACTATGGAA TGAACTGGGTGCGCCAGGCT 120 CCAGGAAAGG GTTTAGAGTG GATGGGCTGG ATAAACACCC ACACTGGAGAGCCAACATAT 180 GCTGATTCTT TCAAGGGACG GTTTACCTTC TCTTTGGACG ATTCTAAGAACACTGCCTAT 240 TTACAGATCA ACAGCCTCAG AGCCGAGGAC ACGGCTGTGT ATTTCTGTACAAGACGGGGT 300 TACGACTGGT ACTTCGATGT CTGGGGCCAA GGGACCACGG TCACCGTCTCCTCAGGTGGC 360 GGTGGATCTG GTGGAGGTGG GTCCGGAGGT GGAGGATCTG ACATCCAGATGACTCAGTCT 420 CCATCTTCCC TGTCTGCATC TGTAGGAGAC AGAGTCACTA TCACTTGCCGGGCGAGTCAG 480 GACATTAATA GCTATTTAAG CTGGTTCCAG CAGAAACCAG GGAAAGCTCCTAAGACCCTG 540 ATCTATCGTG CAAACAGATT GGAATCTGGG GTCCCATCAA GGTTCAGTGGCAGTGGATCT 600 GGGACAGATT ATACTCTCAC CATCAGCAGC CTGCAATATG AAGATTTTGGAATTTATTAT 660 TGTCAACAGT ATGATGAGTC TCCGTGGACG TTCGGTGGAG GCACCAAGCTTGAGATGAAA 720 TGA 723 51 base pairs nucleic acid single linear DNA 91CGGACCCACC TCCACCAGAT CCACCGCCAC CTTTCATCTC AAGCTTGGTG C 51 19 basepairs nucleic acid single linear DNA 92 GACATCCAGA TGACTCAGT 19 49 basepairs nucleic acid single linear DNA 93 GGTGGAGGTG GGTCCGGAGG TGGAGGATCTGAGATCCAGT TGGTGCAGT 49 35 base pairs nucleic acid single linear DNA 94TGTACTCGAG CCCATCATGA GGAGACGGTG ACCGT 35 49 base pairs nucleic acidsingle linear DNA 95 GGTGGAGGTG GGTCCGGAGG TGGAGGATCT GACATCCAGATGACTCAGT 49 37 base pairs nucleic acid single linear DNA 96 TGTACTCGAGCCCATCATTT CATCTCAAGC TTGGTGC 37 22 base pairs nucleic acid singlelinear DNA 97 GAGATCCAGT TGGTGCAGTC TG 22 49 base pairs nucleic acidsingle linear DNA 98 CGGACCCACC TCCACCAGAT CCACCGCCAC CTGAGGAGACGGTGACCGT 49 251 amino acids amino acid linear protein 99 Gly Leu AspThr Val Ser Phe Ser Thr Lys Gly Ala Thr Tyr Ile Thr 1 5 10 15 Tyr ValAsn Phe Leu Asn Glu Leu Arg Val Lys Leu Lys Pro Glu Gly 20 25 30 Asn SerHis Gly Ile Pro Leu Leu Arg Lys Lys Cys Asp Asp Pro Gly 35 40 45 Lys AlaPhe Val Leu Val Ala Leu Ser Asn Asp Asn Gly Gln Leu Ala 50 55 60 Glu IleAla Ile Asp Val Thr Ser Val Tyr Val Val Gly Tyr Gln Val 65 70 75 80 ArgAsn Arg Ser Tyr Phe Phe Lys Asp Ala Pro Asp Ala Ala Tyr Glu 85 90 95 GlyLeu Phe Lys Asn Thr Ile Lys Thr Arg Leu His Phe Gly Gly Ser 100 105 110Tyr Pro Ser Leu Glu Gly Glu Lys Ala Tyr Arg Glu Thr Thr Asp Leu 115 120125 Gly Ile Glu Pro Leu Arg Ile Gly Ile Lys Lys Leu Asp Glu Asn Ala 130135 140 Ile Asp Asn Tyr Lys Pro Thr Glu Ile Ala Ser Ser Leu Leu Val Val145 150 155 160 Ile Gln Met Val Ser Glu Ala Ala Arg Phe Thr Phe Ile GluAsn Gln 165 170 175 Ile Arg Asn Asn Phe Gln Gln Arg Ile Arg Pro Ala AsnAsn Thr Ile 180 185 190 Ser Leu Glu Asn Lys Trp Gly Lys Leu Ser Phe GlnIle Arg Thr Ser 195 200 205 Gly Ala Asn Gly Met Phe Ser Glu Ala Val GluLeu Glu Arg Ala Asn 210 215 220 Gly Lys Lys Tyr Tyr Val Thr Ala Val AspGln Val Lys Pro Lys Ile 225 230 235 240 Ala Leu Leu Lys Phe Val Asp LysAsp Pro Lys 245 250 251 amino acids amino acid linear protein 100 GlyLeu Asp Thr Val Ser Phe Ser Thr Lys Gly Ala Thr Tyr Ile Thr 1 5 10 15Tyr Val Asn Phe Leu Asn Glu Leu Arg Val Lys Leu Lys Pro Glu Gly 20 25 30Asn Ser His Gly Ile Pro Leu Leu Arg Lys Lys Ala Asp Asp Pro Gly 35 40 45Lys Cys Phe Val Leu Val Ala Leu Ser Asn Asp Asn Gly Gln Leu Ala 50 55 60Glu Ile Ala Ile Asp Val Thr Ser Val Tyr Val Val Gly Tyr Gln Val 65 70 7580 Arg Asn Arg Ser Tyr Phe Phe Lys Asp Ala Pro Asp Ala Ala Tyr Glu 85 9095 Gly Leu Phe Lys Asn Thr Ile Lys Thr Arg Leu His Phe Gly Gly Ser 100105 110 Tyr Pro Ser Leu Glu Gly Glu Lys Ala Tyr Arg Glu Thr Thr Asp Leu115 120 125 Gly Ile Glu Pro Leu Arg Ile Gly Ile Lys Lys Leu Asp Glu AsnAla 130 135 140 Ile Asp Asn Tyr Lys Pro Thr Glu Ile Ala Ser Ser Leu LeuVal Val 145 150 155 160 Ile Gln Met Val Ser Glu Ala Ala Arg Phe Thr PheIle Glu Asn Gln 165 170 175 Ile Arg Asn Asn Phe Gln Gln Arg Ile Arg ProAla Asn Asn Thr Ile 180 185 190 Ser Leu Glu Asn Lys Trp Gly Lys Leu SerPhe Gln Ile Arg Thr Ser 195 200 205 Gly Ala Asn Gly Met Phe Ser Glu AlaVal Glu Leu Glu Arg Ala Asn 210 215 220 Gly Lys Lys Tyr Tyr Val Thr AlaVal Asp Gln Val Lys Pro Lys Ile 225 230 235 240 Ala Leu Leu Lys Phe ValAsp Lys Asp Pro Lys 245 250 251 amino acids amino acid linear protein101 Gly Leu Asp Thr Val Ser Phe Ser Thr Lys Gly Ala Thr Tyr Ile Thr 1 510 15 Tyr Val Asn Phe Leu Asn Glu Leu Arg Val Lys Leu Lys Pro Glu Gly 2025 30 Asn Ser His Gly Ile Pro Leu Leu Arg Lys Lys Ala Asp Asp Pro Gly 3540 45 Lys Ala Phe Val Leu Val Ala Leu Ser Asn Asp Asn Gly Gln Leu Ala 5055 60 Glu Ile Ala Ile Asp Val Thr Ser Val Tyr Val Val Gly Tyr Gln Val 6570 75 80 Arg Asn Arg Ser Tyr Phe Phe Lys Asp Ala Pro Asp Ala Ala Tyr Glu85 90 95 Gly Leu Phe Lys Asn Thr Ile Lys Thr Arg Leu His Phe Gly Gly Ser100 105 110 Tyr Pro Ser Leu Glu Gly Glu Lys Ala Tyr Arg Glu Thr Thr AspLeu 115 120 125 Gly Ile Glu Pro Leu Arg Ile Gly Ile Lys Lys Leu Asp GluAsn Ala 130 135 140 Ile Asp Asn Tyr Lys Pro Thr Glu Ile Ala Ser Ser LeuLeu Val Val 145 150 155 160 Ile Gln Met Val Ser Glu Ala Ala Arg Phe ThrPhe Ile Glu Asn Gln 165 170 175 Ile Arg Asn Asn Phe Gln Gln Arg Ile ArgPro Ala Asn Asn Thr Ile 180 185 190 Ser Leu Glu Asn Lys Trp Gly Lys LeuSer Phe Gln Ile Arg Thr Ser 195 200 205 Gly Ala Asn Gly Met Phe Ser GluAla Val Glu Leu Glu Arg Ala Asn 210 215 220 Gly Lys Lys Tyr Tyr Val ThrAla Val Asp Gln Val Lys Pro Lys Ile 225 230 235 240 Ala Leu Leu Lys PheVal Asp Lys Asp Pro Lys 245 250 251 amino acids amino acid linearprotein 102 Gly Leu Asp Thr Val Ser Phe Ser Thr Lys Gly Ala Thr Tyr IleThr 1 5 10 15 Tyr Val Asn Phe Leu Asn Glu Leu Arg Val Lys Leu Lys ProGlu Gly 20 25 30 Asn Ser His Gly Ile Pro Leu Leu Arg Lys Lys Cys Asp AspPro Gly 35 40 45 Lys Cys Phe Val Leu Val Ala Leu Ser Asn Asp Asn Gly GlnLeu Ala 50 55 60 Glu Ile Ala Ile Asp Val Thr Ser Val Tyr Val Val Gly TyrGln Val 65 70 75 80 Arg Asn Arg Ser Tyr Phe Phe Lys Asp Ala Pro Asp AlaAla Tyr Glu 85 90 95 Gly Leu Phe Lys Asn Thr Ile Lys Thr Arg Leu His PheGly Gly Ser 100 105 110 Tyr Pro Ser Leu Glu Gly Glu Lys Ala Tyr Arg GluThr Thr Asp Leu 115 120 125 Gly Ile Glu Pro Leu Arg Ile Gly Ile Lys LysLeu Asp Glu Asn Ala 130 135 140 Ile Asp Asn Tyr Lys Pro Thr Glu Ile AlaSer Ser Leu Leu Val Val 145 150 155 160 Ile Gln Met Val Ser Glu Ala AlaArg Phe Thr Phe Ile Glu Asn Gln 165 170 175 Ile Arg Asn Asn Phe Gln GlnArg Ile Arg Pro Ala Asn Asn Thr Ile 180 185 190 Ser Leu Glu Asn Lys TrpGly Lys Leu Ser Phe Gln Ile Arg Thr Ser 195 200 205 Gly Ala Asn Gly MetPhe Ser Glu Ala Val Glu Leu Glu Arg Ala Asn 210 215 220 Gly Lys Lys TyrTyr Val Thr Ala Val Asp Gln Val Lys Pro Lys Ile 225 230 235 240 Ala LeuLeu Lys Phe Val Cys Lys Asp Pro Lys 245 250 251 amino acids amino acidlinear protein 103 Gly Leu Asp Thr Val Ser Phe Ser Thr Lys Gly Ala ThrTyr Ile Thr 1 5 10 15 Tyr Val Asn Phe Leu Asn Glu Leu Arg Val Lys LeuLys Pro Glu Gly 20 25 30 Asn Ser His Gly Ile Pro Leu Leu Arg Lys Lys CysAsp Asp Pro Gly 35 40 45 Lys Cys Phe Val Leu Val Ala Leu Ser Asn Asp AsnGly Gln Leu Ala 50 55 60 Glu Ile Ala Ile Asp Val Thr Ser Val Tyr Val ValGly Tyr Gln Val 65 70 75 80 Arg Asn Arg Ser Tyr Phe Phe Lys Asp Ala ProAsp Ala Ala Tyr Glu 85 90 95 Gly Leu Phe Lys Asn Thr Ile Lys Thr Arg LeuHis Phe Gly Gly Ser 100 105 110 Tyr Pro Ser Leu Glu Gly Glu Lys Ala TyrArg Glu Thr Thr Asp Leu 115 120 125 Gly Ile Glu Pro Leu Arg Ile Gly IleLys Lys Leu Asp Glu Asn Ala 130 135 140 Ile Asp Asn Tyr Lys Pro Thr GluIle Ala Ser Ser Leu Leu Val Val 145 150 155 160 Ile Gln Met Val Ser GluAla Ala Arg Phe Thr Phe Ile Glu Asn Gln 165 170 175 Ile Arg Asn Asn PheGln Gln Arg Ile Arg Pro Ala Asn Asn Thr Ile 180 185 190 Ser Leu Glu AsnLys Trp Gly Lys Leu Ser Phe Gln Ile Arg Thr Ser 195 200 205 Gly Ala AsnGly Met Phe Ser Glu Ala Val Glu Leu Glu Arg Ala Asn 210 215 220 Gly LysLys Tyr Tyr Val Thr Ala Val Asp Gln Val Lys Pro Lys Ile 225 230 235 240Ala Leu Leu Lys Phe Val Asp Cys Asp Pro Lys 245 250 251 amino acidsamino acid linear protein 104 Gly Leu Asp Thr Val Ser Phe Ser Thr LysGly Ala Thr Tyr Ile Thr 1 5 10 15 Tyr Val Asn Phe Leu Asn Glu Leu ArgVal Lys Leu Lys Pro Glu Gly 20 25 30 Asn Ser His Gly Ile Pro Leu Leu ArgLys Lys Cys Asp Asp Pro Gly 35 40 45 Lys Cys Phe Val Leu Val Ala Leu SerAsn Asp Asn Gly Gln Leu Ala 50 55 60 Glu Ile Ala Ile Asp Val Thr Ser ValTyr Val Val Gly Tyr Gln Val 65 70 75 80 Arg Asn Arg Ser Tyr Phe Phe LysAsp Ala Pro Asp Ala Ala Tyr Glu 85 90 95 Gly Leu Phe Lys Asn Thr Ile LysThr Arg Leu His Phe Gly Gly Ser 100 105 110 Tyr Pro Ser Leu Glu Gly GluLys Ala Tyr Arg Glu Thr Thr Asp Leu 115 120 125 Gly Ile Glu Pro Leu ArgIle Gly Ile Lys Lys Leu Asp Glu Asn Ala 130 135 140 Ile Asp Asn Tyr LysPro Thr Glu Ile Ala Ser Ser Leu Leu Val Val 145 150 155 160 Ile Gln MetVal Ser Glu Ala Ala Arg Phe Thr Phe Ile Glu Asn Gln 165 170 175 Ile ArgAsn Asn Phe Gln Gln Arg Ile Arg Pro Ala Asn Asn Thr Ile 180 185 190 SerLeu Glu Asn Lys Trp Gly Lys Leu Ser Phe Gln Ile Arg Thr Ser 195 200 205Gly Ala Asn Gly Met Phe Ser Glu Ala Val Glu Leu Glu Arg Ala Asn 210 215220 Gly Lys Lys Tyr Tyr Val Thr Ala Val Asp Gln Val Lys Pro Cys Ile 225230 235 240 Ala Leu Leu Lys Phe Val Asp Lys Asp Pro Lys 245 250 251amino acids amino acid linear protein 105 Gly Leu Asp Thr Val Ser PheSer Thr Lys Gly Ala Thr Tyr Ile Thr 1 5 10 15 Tyr Val Asn Phe Leu AsnGlu Leu Arg Val Lys Leu Lys Pro Glu Gly 20 25 30 Asn Ser His Gly Ile ProLeu Leu Arg Lys Lys Cys Asp Asp Pro Gly 35 40 45 Lys Cys Phe Val Leu ValAla Leu Ser Asn Asp Asn Gly Gln Leu Ala 50 55 60 Glu Ile Ala Ile Asp ValThr Ser Val Tyr Val Val Gly Tyr Gln Val 65 70 75 80 Arg Asn Arg Ser TyrPhe Phe Lys Asp Ala Pro Asp Ala Ala Tyr Glu 85 90 95 Gly Leu Phe Lys AsnThr Ile Lys Thr Arg Leu His Phe Gly Gly Ser 100 105 110 Tyr Pro Ser LeuGlu Gly Glu Lys Ala Tyr Arg Glu Thr Thr Asp Leu 115 120 125 Gly Ile GluPro Leu Arg Ile Gly Ile Lys Lys Leu Asp Glu Asn Ala 130 135 140 Ile AspAsn Tyr Lys Pro Thr Glu Ile Ala Ser Ser Leu Leu Val Val 145 150 155 160Ile Gln Met Val Ser Glu Ala Ala Arg Phe Thr Phe Ile Glu Asn Gln 165 170175 Ile Arg Asn Asn Phe Gln Gln Arg Ile Arg Pro Ala Asn Asn Thr Ile 180185 190 Ser Leu Glu Asn Lys Trp Gly Lys Leu Ser Phe Gln Ile Arg Thr Ser195 200 205 Gly Ala Asn Gly Met Phe Ser Glu Ala Val Glu Leu Glu Arg AlaAsn 210 215 220 Gly Lys Lys Tyr Tyr Val Thr Ala Val Asp Gln Val Lys ProLys Ile 225 230 235 240 Ala Leu Leu Cys Phe Val Asp Lys Asp Pro Lys 245250 251 amino acids amino acid linear protein 106 Gly Leu Asp Thr ValSer Phe Ser Thr Cys Gly Ala Thr Tyr Ile Thr 1 5 10 15 Tyr Val Asn PheLeu Asn Glu Leu Arg Val Lys Leu Lys Pro Glu Gly 20 25 30 Asn Ser His GlyIle Pro Leu Leu Arg Lys Lys Cys Asp Asp Pro Gly 35 40 45 Lys Cys Phe ValLeu Val Ala Leu Ser Asn Asp Asn Gly Gln Leu Ala 50 55 60 Glu Ile Ala IleAsp Val Thr Ser Val Tyr Val Val Gly Tyr Gln Val 65 70 75 80 Arg Asn ArgSer Tyr Phe Phe Lys Asp Ala Pro Asp Ala Ala Tyr Glu 85 90 95 Gly Leu PheLys Asn Thr Ile Lys Thr Arg Leu His Phe Gly Gly Ser 100 105 110 Tyr ProSer Leu Glu Gly Glu Lys Ala Tyr Arg Glu Thr Thr Asp Leu 115 120 125 GlyIle Glu Pro Leu Arg Ile Gly Ile Lys Lys Leu Asp Glu Asn Ala 130 135 140Ile Asp Asn Tyr Lys Pro Thr Glu Ile Ala Ser Ser Leu Leu Val Val 145 150155 160 Ile Gln Met Val Ser Glu Ala Ala Arg Phe Thr Phe Ile Glu Asn Gln165 170 175 Ile Arg Asn Asn Phe Gln Gln Arg Ile Arg Pro Ala Asn Asn ThrIle 180 185 190 Ser Leu Glu Asn Lys Trp Gly Lys Leu Ser Phe Gln Ile ArgThr Ser 195 200 205 Gly Ala Asn Gly Met Phe Ser Glu Ala Val Glu Leu GluArg Ala Asn 210 215 220 Gly Lys Lys Tyr Tyr Val Thr Ala Val Asp Gln ValLys Pro Lys Ile 225 230 235 240 Ala Leu Leu Lys Phe Val Asp Lys Asp ProLys 245 250 251 amino acids amino acid linear protein 107 Gly Leu AspThr Val Ser Phe Ser Thr Lys Gly Ala Thr Tyr Ile Thr 1 5 10 15 Tyr ValAsn Phe Leu Asn Glu Leu Arg Val Lys Leu Lys Pro Glu Gly 20 25 30 Asn SerHis Gly Ile Pro Leu Leu Arg Lys Lys Cys Asp Asp Pro Gly 35 40 45 Lys CysPhe Val Leu Val Ala Leu Ser Asn Asp Cys Gly Gln Leu Ala 50 55 60 Glu IleAla Ile Asp Val Thr Ser Val Tyr Val Val Gly Tyr Gln Val 65 70 75 80 ArgAsn Arg Ser Tyr Phe Phe Lys Asp Ala Pro Asp Ala Ala Tyr Glu 85 90 95 GlyLeu Phe Lys Asn Thr Ile Lys Thr Arg Leu His Phe Gly Gly Ser 100 105 110Tyr Pro Ser Leu Glu Gly Glu Lys Ala Tyr Arg Glu Thr Thr Asp Leu 115 120125 Gly Ile Glu Pro Leu Arg Ile Gly Ile Lys Lys Leu Asp Glu Asn Ala 130135 140 Ile Asp Asn Tyr Lys Pro Thr Glu Ile Ala Ser Ser Leu Leu Val Val145 150 155 160 Ile Gln Met Val Ser Glu Ala Ala Arg Phe Thr Phe Ile GluAsn Gln 165 170 175 Ile Arg Asn Asn Phe Gln Gln Arg Ile Arg Pro Ala AsnAsn Thr Ile 180 185 190 Ser Leu Glu Asn Lys Trp Gly Lys Leu Ser Phe GlnIle Arg Thr Ser 195 200 205 Gly Ala Asn Gly Met Phe Ser Glu Ala Val GluLeu Glu Arg Ala Asn 210 215 220 Gly Lys Lys Tyr Tyr Val Thr Ala Val AspGln Val Lys Pro Lys Ile 225 230 235 240 Ala Leu Leu Lys Phe Val Asp LysAsp Pro Lys 245 250 251 amino acids amino acid linear protein 108 GlyLeu Asp Thr Val Ser Phe Ser Thr Lys Gly Ala Thr Tyr Ile Thr 1 5 10 15Tyr Val Asn Phe Leu Asn Glu Leu Arg Val Lys Leu Lys Pro Glu Gly 20 25 30Asn Ser His Gly Ile Pro Leu Leu Arg Lys Lys Cys Asp Asp Pro Gly 35 40 45Lys Cys Phe Val Leu Val Ala Leu Ser Asn Asp Asn Gly Gln Leu Ala 50 55 60Glu Ile Ala Ile Asp Val Thr Ser Val Tyr Val Val Gly Tyr Gln Val 65 70 7580 Arg Asn Arg Ser Tyr Phe Phe Lys Asp Ala Pro Asp Ala Ala Tyr Glu 85 9095 Gly Leu Phe Lys Asn Thr Cys Lys Thr Arg Leu His Phe Gly Gly Ser 100105 110 Tyr Pro Ser Leu Glu Gly Glu Lys Ala Tyr Arg Glu Thr Thr Asp Leu115 120 125 Gly Ile Glu Pro Leu Arg Ile Gly Ile Lys Lys Leu Asp Glu AsnAla 130 135 140 Ile Asp Asn Tyr Lys Pro Thr Glu Ile Ala Ser Ser Leu LeuVal Val 145 150 155 160 Ile Gln Met Val Ser Glu Ala Ala Arg Phe Thr PheIle Glu Asn Gln 165 170 175 Ile Arg Asn Asn Phe Gln Gln Arg Ile Arg ProAla Asn Asn Thr Ile 180 185 190 Ser Leu Glu Asn Lys Trp Gly Lys Leu SerPhe Gln Ile Arg Thr Ser 195 200 205 Gly Ala Asn Gly Met Phe Ser Glu AlaVal Glu Leu Glu Arg Ala Asn 210 215 220 Gly Lys Lys Tyr Tyr Val Thr AlaVal Asp Gln Val Lys Pro Lys Ile 225 230 235 240 Ala Leu Leu Lys Phe ValAsp Lys Asp Pro Lys 245 250 251 amino acids amino acid linear protein109 Gly Leu Asp Thr Val Ser Phe Ser Thr Lys Gly Ala Thr Tyr Ile Thr 1 510 15 Tyr Val Asn Phe Leu Asn Glu Leu Arg Val Lys Leu Lys Pro Glu Gly 2025 30 Asn Ser His Gly Ile Pro Leu Leu Arg Lys Lys Cys Asp Asp Pro Gly 3540 45 Lys Cys Phe Val Leu Val Ala Leu Ser Asn Asp Asn Gly Gln Leu Ala 5055 60 Glu Ile Ala Ile Asp Val Thr Ser Val Tyr Val Val Gly Tyr Gln Val 6570 75 80 Arg Asn Arg Ser Tyr Phe Phe Lys Asp Ala Pro Asp Ala Ala Tyr Glu85 90 95 Gly Leu Phe Lys Asn Thr Ile Lys Thr Arg Leu His Phe Gly Gly Ser100 105 110 Tyr Pro Ser Leu Glu Gly Glu Lys Ala Tyr Arg Glu Thr Thr AspLeu 115 120 125 Gly Ile Glu Pro Leu Arg Ile Gly Ile Lys Lys Leu Asp GluAsn Ala 130 135 140 Ile Asp Asn Tyr Lys Pro Thr Glu Ile Ala Ser Ser LeuLeu Val Val 145 150 155 160 Ile Gln Met Val Ser Glu Ala Ala Arg Phe ThrPhe Ile Glu Asn Gln 165 170 175 Ile Arg Asn Asn Phe Gln Gln Cys Ile ArgPro Ala Asn Asn Thr Ile 180 185 190 Ser Leu Glu Asn Lys Trp Gly Lys LeuSer Phe Gln Ile Arg Thr Ser 195 200 205 Gly Ala Asn Gly Met Phe Ser GluAla Val Glu Leu Glu Arg Ala Asn 210 215 220 Gly Lys Lys Tyr Tyr Val ThrAla Val Asp Gln Val Lys Pro Lys Ile 225 230 235 240 Ala Leu Leu Lys PheVal Asp Lys Asp Pro Lys 245 250 251 amino acids amino acid linearprotein 110 Gly Leu Asp Thr Val Ser Phe Ser Thr Cys Gly Ala Thr Tyr IleThr 1 5 10 15 Tyr Val Asn Phe Leu Asn Glu Leu Arg Val Lys Leu Lys ProGlu Gly 20 25 30 Asn Ser His Gly Ile Pro Leu Leu Arg Lys Lys Ala Asp AspPro Gly 35 40 45 Lys Ala Phe Val Leu Val Ala Leu Ser Asn Asp Asn Gly GlnLeu Ala 50 55 60 Glu Ile Ala Ile Asp Val Thr Ser Val Tyr Val Val Gly TyrGln Val 65 70 75 80 Arg Asn Arg Ser Tyr Phe Phe Lys Asp Ala Pro Asp AlaAla Tyr Glu 85 90 95 Gly Leu Phe Lys Asn Thr Ile Lys Thr Arg Leu His PheGly Gly Ser 100 105 110 Tyr Pro Ser Leu Glu Gly Glu Lys Ala Tyr Arg GluThr Thr Asp Leu 115 120 125 Gly Ile Glu Pro Leu Arg Ile Gly Ile Lys LysLeu Asp Glu Asn Ala 130 135 140 Ile Asp Asn Tyr Lys Pro Thr Glu Ile AlaSer Ser Leu Leu Val Val 145 150 155 160 Ile Gln Met Val Ser Glu Ala AlaArg Phe Thr Phe Ile Glu Asn Gln 165 170 175 Ile Arg Asn Asn Phe Gln GlnArg Ile Arg Pro Ala Asn Asn Thr Ile 180 185 190 Ser Leu Glu Asn Lys TrpGly Lys Leu Ser Phe Gln Ile Arg Thr Ser 195 200 205 Gly Ala Asn Gly MetPhe Ser Glu Ala Val Glu Leu Glu Arg Ala Asn 210 215 220 Gly Lys Lys TyrTyr Val Thr Ala Val Asp Gln Val Lys Pro Lys Ile 225 230 235 240 Ala LeuLeu Lys Phe Val Asp Lys Asp Pro Lys 245 250 251 amino acids amino acidlinear protein 111 Gly Leu Asp Thr Val Ser Phe Ser Thr Cys Gly Ala ThrTyr Ile Thr 1 5 10 15 Tyr Val Asn Phe Leu Asn Glu Leu Arg Val Lys LeuLys Pro Glu Gly 20 25 30 Asn Ser His Gly Ile Pro Leu Leu Arg Lys Lys AlaAsp Asp Pro Gly 35 40 45 Lys Ala Phe Val Leu Val Ala Leu Ser Asn Asp AsnGly Gln Leu Ala 50 55 60 Glu Ile Ala Ile Asp Val Thr Ser Val Tyr Val ValGly Tyr Gln Val 65 70 75 80 Arg Asn Arg Ser Tyr Phe Phe Lys Asp Ala ProAsp Ala Ala Tyr Glu 85 90 95 Gly Leu Phe Lys Asn Thr Ile Lys Thr Arg LeuHis Phe Gly Gly Ser 100 105 110 Tyr Pro Ser Leu Glu Gly Glu Lys Ala TyrArg Glu Thr Thr Asp Leu 115 120 125 Gly Ile Glu Pro Leu Arg Ile Gly IleLys Lys Leu Asp Glu Asn Ala 130 135 140 Ile Asp Asn Tyr Lys Pro Thr GluIle Ala Ser Ser Leu Leu Val Val 145 150 155 160 Ile Gln Met Val Ser GluAla Ala Arg Phe Thr Phe Ile Glu Asn Gln 165 170 175 Ile Arg Asn Asn PheGln Gln Arg Ile Arg Pro Ala Asn Asn Thr Ile 180 185 190 Ser Leu Glu AsnLys Trp Gly Lys Leu Ser Phe Gln Ile Arg Thr Ser 195 200 205 Gly Ala AsnGly Met Phe Ser Glu Ala Val Glu Leu Glu Arg Ala Asn 210 215 220 Gly LysLys Tyr Tyr Val Thr Ala Val Asp Gln Val Lys Pro Lys Ile 225 230 235 240Ala Leu Leu Lys Phe Val Cys Lys Asp Pro Lys 245 250 29 base pairsnucleic acid single linear DNA 112 TGATGCGGCC GACATCTCAA GCTTGGTGC 29 29base pairs nucleic acid single linear DNA 113 TGATGCGGCC GACATCTCAAGCTTGGTGC 29 38 base pairs nucleic acid single linear DNA 114 TCTAGGTCACCGTCTCCTCA CCATCTGGAC AGGCTGGA 38 37 base pairs nucleic acid singlelinear DNA 115 TTCGAAGCTT GAGATGAAAC CATCTGGACA GGCTGGA 37 27 base pairsnucleic acid single linear DNA 116 AGTCGTCGAC ACGATGGACA TGAGGAC 27 98base pairs nucleic acid single linear DNA 117 AGTCGTCGAC ACGATGGACATGAGGACCCC TGCTCAGTTT CTTGGCATCC TCCTACTCTG 60 GTTTCCAGGT ATCAAATGTGACATCCAGAT GACTCAGT 98 79 base pairs nucleic acid single linear DNA 118TCACTTGCCG GGCGAATCAG GACATTAATA GCTATTTAAG CTGGTTCCAG CAGAAACCAG 60GGAAAGCTCC TAAGACCCT 79 80 base pairs nucleic acid single linear DNA 119TGACTCGCCC GGCAAGTGAT AGTGACTCTG TCTCCTACAG ATGCAGACAG GGAAGATGGA 60GACTGAGTCA TCTGGATGTC 80 79 base pairs nucleic acid single linear DNA120 GATCCACTGC CACTGAACCT TGATGGGACC CCAGATTCCA ATCTGTTTGC ACGATAGATC 60AGGGTCTTAG GAGCTTTCC 79 82 base pairs nucleic acid single linear DNA 121GGTTCAGTGG CAGTGGATCT GGGACAGATT ATACTCTCAC CATCAGCAGC CTGCAATATG 60AAGATTTTGG AATTTATTAT TG 82 82 base pairs nucleic acid single linear DNA122 GTTTGATTTC AAGCTTGGTG CCTCCACCGA ACGTCCACGG AGACTCATCA TACTGTTGAC 60AATAATAAAT TCCAAAATCT TC 82 107 amino acids amino acid single linearprotein 123 Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser LeuGly 1 5 10 15 Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile AsnSer Tyr 20 25 30 Leu Ser Trp Phe His His Lys Pro Gly Lys Ser Pro Lys ThrLeu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg PheSer Gly 50 55 60 Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser LeuAsp Tyr 65 70 75 80 Glu Asp Met Gly Ile Tyr Tyr Cys Gln Gln Tyr Asp GluSer Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105118 amino acids amino acid single linear protein 124 Gln Ile Gln Leu ValGln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys IleSer Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn TrpVal Lys Gln Ala Pro Gly Lys Gly Leu Arg Trp Met 35 40 45 Gly Trp Ile AsnThr His Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg PheAla Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln IleAsn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Thr Arg ArgGly Tyr Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr 100 105 110 Thr ValThr Val Ser Ser 115 107 amino acids amino acid single linear protein 125Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 1015 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Ser Tyr 20 2530 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile 35 4045 Tyr Arg Ala Asn Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 5560 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Tyr 65 7075 80 Glu Asp Phe Gly Ile Tyr Tyr Cys Gln Gln Tyr Asp Glu Ser Pro Trp 8590 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 118 aminoacids amino acid single linear protein 126 Glu Ile Gln Leu Val Gln SerGly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Val Arg Ile Ser CysAla Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val ArgGln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr HisThr Gly Glu Pro Thr Tyr Ala Asp Ser Phe 50 55 60 Lys Gly Arg Phe Thr PheSer Leu Asp Asp Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn SerLeu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Thr Arg Arg Gly TyrAsp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr ValSer Ser 115 280 amino acids amino acid linear protein 127 Ala Ala LysMet Ala Lys Asn Val Asp Lys Pro Leu Phe Thr Ala Thr 1 5 10 15 Phe AsnVal Gln Ala Ser Ser Ala Asp Tyr Ala Thr Phe Ile Ala Gly 20 25 30 Ile ArgAsn Lys Leu Arg Asn Pro Ala His Phe Ser His Asn Arg Pro 35 40 45 Val LeuPro Pro Val Glu Pro Asn Val Pro Pro Ser Arg Trp Phe His 50 55 60 Val ValLeu Lys Ala Ser Pro Thr Ser Ala Gly Leu Thr Leu Ala Ile 65 70 75 80 ArgAla Asp Asn Ile Tyr Leu Glu Gly Phe Lys Ser Ser Asp Gly Thr 85 90 95 TrpTrp Glu Leu Thr Pro Gly Leu Ile Pro Gly Ala Thr Tyr Val Gly 100 105 110Phe Gly Gly Thr Tyr Arg Asp Leu Leu Gly Asp Thr Asp Lys Leu Thr 115 120125 Asn Val Ala Leu Gly Arg Gln Gln Leu Ala Asp Ala Val Thr Ala Leu 130135 140 His Gly Arg Thr Lys Ala Asp Lys Ala Ser Gly Pro Lys Gln Gln Gln145 150 155 160 Ala Arg Glu Ala Val Thr Thr Leu Val Leu Met Val Asn GluAla Thr 165 170 175 Arg Phe Gln Thr Val Ser Gly Phe Val Ala Gly Leu LeuHis Pro Lys 180 185 190 Ala Val Glu Lys Lys Ser Gly Lys Ile Gly Asn GluMet Lys Ala Gln 195 200 205 Val Asn Gly Trp Gln Asp Leu Ser Ala Ala LeuLeu Lys Thr Asp Val 210 215 220 Lys Pro Pro Pro Gly Lys Ser Pro Ala LysPhe Ala Pro Ile Glu Lys 225 230 235 240 Met Gly Val Arg Thr Ala Glu GlnAla Ala Asn Thr Leu Gly Ile Leu 245 250 255 Leu Phe Val Glu Val Pro GlyGly Leu Thr Val Ala Lys Ala Leu Glu 260 265 270 Leu Phe His Ala Cys GlyGly Lys 275 280 280 amino acids amino acid linear protein 128 Ala AlaLys Met Ala Lys Asn Val Asp Lys Pro Leu Phe Thr Ala Thr 1 5 10 15 PheAsn Val Gln Ala Ser Ser Ala Asp Tyr Ala Thr Phe Ile Ala Gly 20 25 30 IleArg Asn Lys Leu Arg Asn Pro Ala His Phe Ser His Asn Arg Pro 35 40 45 ValLeu Pro Pro Val Glu Pro Asn Val Pro Pro Ser Arg Trp Phe His 50 55 60 ValVal Leu Lys Ala Ser Pro Thr Ser Ala Gly Leu Thr Leu Ala Ile 65 70 75 80Arg Ala Asp Asn Ile Tyr Leu Glu Gly Phe Lys Ser Ser Asp Gly Thr 85 90 95Trp Trp Glu Leu Thr Pro Gly Leu Ile Pro Gly Ala Thr Tyr Val Gly 100 105110 Phe Gly Gly Thr Tyr Arg Asp Leu Leu Gly Asp Thr Asp Lys Leu Thr 115120 125 Asn Val Ala Leu Gly Arg Gln Gln Leu Ala Asp Ala Val Thr Ala Leu130 135 140 His Gly Arg Thr Lys Ala Asp Lys Ala Ser Gly Pro Lys Gln GlnGln 145 150 155 160 Ala Arg Glu Ala Val Thr Thr Leu Val Leu Met Val AsnGlu Ala Thr 165 170 175 Arg Phe Gln Thr Val Ser Gly Phe Val Ala Gly LeuLeu His Pro Lys 180 185 190 Ala Val Glu Lys Lys Ser Gly Lys Ile Gly AsnGlu Met Lys Ala Gln 195 200 205 Val Asn Gly Trp Gln Asp Leu Ser Ala AlaLeu Leu Lys Thr Asp Val 210 215 220 Lys Pro Pro Pro Gly Lys Ser Pro AlaLys Phe Ala Pro Ile Glu Lys 225 230 235 240 Met Gly Val Arg Thr Ala GluGln Ala Ala Asn Thr Leu Gly Ile Leu 245 250 255 Leu Phe Val Glu Val ProGly Gly Leu Thr Val Ala Lys Cys Leu Glu 260 265 270 Leu Phe His Ala SerGly Gly Lys 275 280 280 amino acids amino acid linear protein 129 AlaAla Lys Met Ala Lys Asn Val Asp Lys Pro Leu Phe Thr Ala Thr 1 5 10 15Phe Asn Val Gln Ala Ser Ser Ala Asp Tyr Ala Thr Phe Ile Ala Gly 20 25 30Ile Arg Asn Lys Leu Arg Asn Pro Ala His Phe Ser His Asn Arg Pro 35 40 45Val Leu Pro Pro Val Glu Pro Asn Val Pro Pro Ser Arg Trp Phe His 50 55 60Val Val Leu Lys Ala Ser Pro Thr Ser Ala Gly Leu Thr Leu Ala Ile 65 70 7580 Arg Ala Asp Asn Ile Tyr Leu Glu Gly Phe Lys Ser Ser Asp Gly Thr 85 9095 Trp Trp Glu Leu Thr Pro Gly Leu Ile Pro Gly Ala Thr Tyr Val Gly 100105 110 Phe Gly Gly Thr Tyr Arg Asp Leu Leu Gly Asp Thr Asp Lys Leu Thr115 120 125 Asn Val Ala Leu Gly Arg Gln Gln Leu Ala Asp Ala Val Thr AlaLeu 130 135 140 His Gly Arg Thr Lys Ala Asp Lys Ala Ser Gly Pro Lys GlnGln Gln 145 150 155 160 Ala Arg Glu Ala Val Thr Thr Leu Val Leu Met ValAsn Glu Ala Thr 165 170 175 Arg Phe Gln Thr Val Ser Gly Phe Val Ala GlyLeu Leu His Pro Lys 180 185 190 Ala Val Glu Lys Lys Ser Gly Lys Ile GlyAsn Glu Met Lys Ala Gln 195 200 205 Val Asn Gly Trp Gln Asp Leu Ser AlaAla Leu Leu Lys Thr Asp Val 210 215 220 Lys Pro Pro Pro Gly Lys Ser ProAla Lys Phe Ala Pro Ile Glu Lys 225 230 235 240 Met Gly Val Arg Thr AlaGlu Gln Ala Ala Asn Thr Leu Gly Ile Cys 245 250 255 Leu Phe Val Glu ValPro Gly Gly Leu Thr Val Ala Lys Ala Leu Glu 260 265 270 Leu Phe His AlaSer Gly Gly Lys 275 280 7 amino acids amino acid linear protein 130 SerCys Asp Lys Thr His Thr 1 5 85 base pairs nucleic acid single linear DNA131 TGTCGACATC ATGGCTTGGG TGTGGACCTT GCTATTCCTG ATGGCAGCTG CCCAAAGTGC 60CCAAGCAGAG ATCCAGTTGG TGCAG 85 86 base pairs nucleic acid single linearDNA 132 AAGGTATACC CAGAAGCTGC GCAGGAGATT CTGACGGACC CTCCAGGCTTCACCAGGCCT 60 CCTCCAGACT GCACCAACTG GATCTC 86 84 base pairs nucleic acidsingle linear DNA 133 GCAGCTTCTG GGTATACCTT CACAAACTAT GGAATGAACTGGGTGCGCCA GGCTCCAGGA 60 AAGAATTTAG AGTGGATGGG CTGG 84 85 base pairsnucleic acid single linear DNA 134 AAAGAGAAGG TAAACCGTCC CTTGAAAGAATCAGCATATG TTGGCTCTCC AGTGTGGGTG 60 TTTATCCAGC CCATCCACTC TAAAC 85 87base pairs nucleic acid single linear DNA 135 GACGGTTTAC CTTCTCTTTGGACGATTCTA AGAACACTGC CTATTTACAG ATCAACAGCC 60 TCAGAGCCGA GGACACGGCTGTGTATT 87 92 base pairs nucleic acid single linear DNA 136 GAGGAGACGGTGACCGTGGT CCCTTGGCCC CAGACATCGA AGTACCAGTC GTAACCCCGT 60 CTTGTACAGAAATACACAGC CGTGTCCTCG GC 92 84 base pairs nucleic acid single linear DNA137 GCAGCTTCTG GGTATACCTT CACAAACTAT GGAATGAACT GGGTGAAGCA GGCTCCAGGA 60AAGGGTTTAA GGTGGATGGG CTGG 84 85 base pairs nucleic acid single linearDNA 138 AAAGAGAAGG TAAACCGTCC CTTGAAGTCA TCAGCATATG TTGGCTCTCCAGTGTGGGTG 60 TTTATCCAGC CCATCCACCT TAAAC 85 84 base pairs nucleic acidsingle linear DNA 139 GACGGTTTAC CTTCTCTTTG GACACGTCTA AGTGCACTGCCTATTTACAG ATCAACAGCC 60 TCAGAGCCGA GGACACGGCT ACAT 84 91 base pairsnucleic acid single linear DNA 140 AGGAGACGGT GACCGTGGTC CCTTGGCCCCAGACATCGAA GTACCAGTCG TAACCCCGTC 60 TTGTACAGAA ATATGTAGCC GTGTCCTCGG C91 8 amino acids amino acid linear protein 141 Lys Pro Ala Lys Phe PheArg Leu 1 5 8 amino acids amino acid linear protein 142 Lys Pro Ala LysPhe Leu Arg Leu 1 5 34 base pairs nucleic acid single linear DNA 143GGCCGCAAAG CCGGCTAAGT TCTTMCGTCT GAGT 34 30 base pairs nucleic acidsingle linear DNA 144 ACTCAGACGK AAGAACTTAG CCGGCTTTGC 30 85 base pairsnucleic acid single linear DNA 145 AAGGTATACC CAGAAGCTGC GCAGGAGATTCTGACGGACC CTCCAGGCTT CTTCAGGCCA 60 GGTCCAGACT GCACCAACTG GATCT 85 26base pairs nucleic acid single linear DNA 146 ACTAGTGTCG ACATCATGGCTTGGGT 26 240 amino acids amino acid linear protein 147 Asp Ile Gln MetThr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg ValThr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30 Leu Ser TrpPhe Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile 35 40 45 Tyr Arg AlaAsn Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly SerGly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Tyr 65 70 75 80 Glu AspPhe Gly Ile Tyr Tyr Cys Gln Gln Tyr Asp Glu Ser Pro Trp 85 90 95 Thr PheGly Gly Gly Thr Lys Leu Glu Met Lys Gly Gly Gly Gly Ser 100 105 110 GlyGly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Gln Leu Val Gln 115 120 125Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Val Arg Ile Ser Cys 130 135140 Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Arg 145150 155 160 Gln Ala Pro Gly Lys Gly Leu Glu Trp Met Gly Trp Ile Asn ThrHis 165 170 175 Thr Gly Glu Pro Thr Tyr Ala Asp Ser Phe Lys Gly Arg PheThr Phe 180 185 190 Ser Leu Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln IleAsn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Thr ArgArg Gly Tyr Asp 210 215 220 Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr ThrVal Thr Val Ser Ser 225 230 235 240 240 amino acids amino acid linearprotein 148 Glu Ile Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro GlyGly 1 5 10 15 Ser Val Arg Ile Ser Cys Ala Ala Ser Gly Tyr Thr Phe ThrAsn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu GluTrp Met 35 40 45 Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala AspSer Phe 50 55 60 Lys Gly Arg Phe Thr Phe Ser Leu Asp Asp Ser Lys Asn ThrAla Tyr 65 70 75 80 Leu Gln Ile Asn Ser Leu Arg Ala Glu Asp Thr Ala ValTyr Phe Cys 85 90 95 Thr Arg Arg Gly Tyr Asp Trp Tyr Phe Asp Val Trp GlyGln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser GlyGly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Gln Met Thr GlnSer Pro Ser Ser Leu 130 135 140 Ser Ala Ser Val Gly Asp Arg Val Thr IleThr Cys Arg Ala Ser Gln 145 150 155 160 Asp Ile Asn Ser Tyr Leu Ser TrpPhe Gln Gln Lys Pro Gly Lys Ala 165 170 175 Pro Lys Thr Leu Ile Tyr ArgAla Asn Arg Leu Glu Ser Gly Val Pro 180 185 190 Ser Arg Phe Ser Gly SerGly Ser Gly Thr Asp Tyr Thr Leu Thr Ile 195 200 205 Ser Ser Leu Gln TyrGlu Asp Phe Gly Ile Tyr Tyr Cys Gln Gln Tyr 210 215 220 Asp Glu Ser ProTrp Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys 225 230 235 240 107amino acids amino acid single linear 149 Asp Ile Gln Met Thr Gln Ser ProSer Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr CysArg Ala Ser Gln Xaa Ile Ser Xaa Tyr 20 25 30 Leu Xaa Trp Tyr Gln Gln LysPro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Xaa Leu XaaSer Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Xaa PheThr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr TyrTyr Cys Gln Gln Tyr Xaa Xaa Xaa Pro Xaa 85 90 95 Thr Phe Gly Gln Gly ThrLys Val Glu Ile Lys 100 105 108 amino acids amino acid double linearprotein 150 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser ProGly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val SerSer Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro ArgLeu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp ArgPhe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser ArgLeu Glu 65 70 75 80 Pro Gly Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr GlySer Ser Pro 85 90 95 Xaa Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100105 108 amino acids amino acid double linear 151 Asp Ile Val Met Thr GlnSer Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser IleSer Cys Arg Ser Ser Gln Ser Leu Leu Asn Asn 20 25 30 Tyr Leu Asn Trp TyrLeu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu 35 40 45 Ile Tyr Leu Gly SerAsn Arg Ala Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser GlyThr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu 65 70 75 80 Ala Glu Asp ValGly Val Tyr Tyr Cys Met Gln Ala Leu Gln Xaa Pro 85 90 95 Xaa Thr Phe GlyGln Gly Thr Lys Xaa Glu Ile Lys 100 105 106 amino acids amino aciddouble linear protein 152 Xaa Ser Val Leu Thr Gln Pro Pro Ser Ala SerGly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser SerSer Ile Gly Xaa Asn Xaa 20 25 30 Val Xaa Trp Tyr Gln Gln Leu Pro Gly ThrAla Pro Lys Leu Leu Ile 35 40 45 Tyr Asn Asn Arg Pro Ser Gly Val Pro AspArg Phe Ser Gly Ser Lys 50 55 60 Ser Gly Thr Ser Ala Ser Leu Ala Ile SerGly Leu Gln Ser Glu Asp 65 70 75 80 Glu Ala Asp Tyr Tyr Cys Ala Thr TrpAsp Asp Ser Leu Asp Pro Val 85 90 95 Phe Gly Gly Gly Thr Lys Thr Val LeuGly 100 105 104 amino acids amino acid double linear protein 153 Xaa SerAla Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 SerIle Thr Ile Ser Cys Thr Gly Thr Ser Ser Val Gly Tyr Asn Xaa 20 25 30 ValSer Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Ile Tyr 35 40 45 AspVal Arg Pro Ser Gly Val Arg Phe Ser Gly Ser Lys Ser Gly Asn 50 55 60 ThrAla Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp 65 70 75 80Tyr Tyr Cys Ser Ser Tyr Xaa Gly Xaa Xaa Xaa Xaa Val Phe Gly Gly 85 90 95Gly Thr Lys Leu Thr Val Leu Gly 100 100 amino acids amino acid doublelinear protein 154 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val SerPro Gly Gln 1 5 10 15 Thr Ala Ile Thr Cys Ser Gly Asp Xaa Leu Xaa XaaXaa Tyr Val Xaa 20 25 30 Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val LeuVal Ile Tyr Asp 35 40 45 Arg Pro Ser Gly Ile Pro Gln Arg Phe Ser Gly SerSer Thr Thr Ala 50 55 60 Thr Leu Thr Ile Ser Gly Val Gln Ala Asp Glu AlaAsp Tyr Tyr Cys 65 70 75 80 Gln Xaa Trp Asp Xaa Xaa Xaa Val Val Phe GlyGly Gly Thr Lys Leu 85 90 95 Thr Val Leu Gly 100 106 amino acids aminoacid double linear protein 155 Asn Phe Met Leu Thr Gln Pro His Ser ValSer Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Xaa SerXaa Gly Ile Ala Ser Xaa Tyr 20 25 30 Val Gln Trp Tyr Gln Gln Arg Pro GlySer Ala Pro Thr Thr Val Ile 35 40 45 Tyr Glu Asp Asn Arg Pro Ser Gly ValPro Asp Arg Phe Ser Gly Ser 50 55 60 Ser Ser Asn Ser Ala Ser Leu Thr IleSer Gly Leu Lys Thr Glu Asp 65 70 75 80 Glu Ala Asp Tyr Tyr Cys Gln SerTyr Asp Ser Xaa Xaa Trp Val Phe 85 90 95 Gly Gly Gly Thr Lys Leu Thr ValLeu Gly 100 105 107 amino acids amino acid double linear protein 156 AspIle Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Lys Asn 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu 35 40 45Ile Tyr Trp Ala Ser Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 7580 Gln Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro Xaa 85 9095 Thr Phe Gly Gln Gly Thr Lys Xaa Gly Ile Lys 100 105 105 amino acidsamino acid double linear protein 157 Ser Glu Leu Thr Gln Pro Pro Ser ValSer Val Ala Pro Gly Gln Thr 1 5 10 15 Arg Ile Thr Cys Ser Gly Asp XaaLeu Gly Xaa Tyr Asp Ala Xaa Trp 20 25 30 Tyr Gln Gln Lys Pro Gly Gln AlaPro Leu Leu Val Ile Tyr Gly Arg 35 40 45 Asn Arg Pro Ser Gly Ile Pro AspArg Phe Ser Gly Ser Ser Ser Gly 50 55 60 His Thr Ala Ser Leu Thr Ile ThrGly Ala Gln Ala Glu Asp Glu Ala 65 70 75 80 Asp Tyr Tyr Cys Asn Ser ArgAsp Ser Ser Gly Lys Val Leu Phe Gly 85 90 95 Gly Gly Thr Lys Leu Thr ValLeu Gly 100 105 96 amino acids amino acid double linear protein 158 SerAla Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln Ser 1 5 10 15Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Val Gly Xaa Xaa Tyr Val 20 25 30Ser Trp Tyr Gln Gln His Gly Ala Pro Lys Ile Glu Val Arg Pro Ser 35 40 45Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Asn Thr Ala Ser Leu 50 55 60Thr Val Ser Gly Leu Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser 65 70 7580 Tyr Xaa Xaa Xaa Xaa Xaa Phe Val Phe Gly Gly Thr Lys Thr Val Leu 85 9095 119 amino acids amino acid double linear protein 159 Glu Val Gln LeuVal Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu ArgLeu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Xaa Xaa 20 25 30 Xaa Met XaaTrp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Xaa Xaa IleXaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Tyr Ala Asp Ser Val 50 55 60 Lys Gly ArgPhe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu GlnMet Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala ArgXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Gly Gln Gly 100 105 110 ThrLeu Val Thr Val Ser Ser 115 119 amino acids amino acid double linear 160Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Xaa 1 5 1015 Ser Val Xaa Val Ser Cys Lys Xaa Ser Gly Tyr Tyr Phe Xaa Xaa Tyr 20 2530 Xaa Ile Xaa Trp Val Arg Gln Ala Pro Gly Xaa Gly Leu Glu Trp Val 35 4045 Gly Xaa Ile Xaa Pro Xaa Xaa Gly Xaa Thr Xaa Tyr Ala Pro Xaa Phe 50 5560 Gln Gly Arg Val Thr Xaa Thr Arg Asp Xaa Ser Xaa Asn Thr Ala Tyr 65 7075 80 Met Glu Leu Xaa Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 8590 95 Ala Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Gly Gln Gly100 105 110 Thr Leu Val Thr Val Ser Ser 115 117 amino acids amino aciddouble linear protein 161 Xaa Val Thr Leu Xaa Glu Ser Gly Pro Xaa LeuVal Leu Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Val Ser GlyXaa Ser Leu Ser Xaa Xaa 20 25 30 Xaa Val Xaa Trp Ile Arg Gln Pro Pro GlyLys Xaa Leu Glu Trp Leu 35 40 45 Ala Xaa Ile Xaa Xaa Asp Asp Asp Xaa TyrXaa Thr Ser Leu Arg Ser 50 55 60 Arg Leu Thr Ile Ser Lys Asp Thr Ser LysAsn Gln Val Val Leu Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Asp Pro Xaa Asp ThrAla Thr Tyr Tyr Cys Ala Arg 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa AspVal Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 107amino acids amino acid Not Relevant linear protein 162 Asp Ile Gln MetThr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg ValThr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Thr Trp 20 25 30 Leu Ala TrpTyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Met 35 40 45 Tyr Lys AlaSer Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ile Gly 50 55 60 Ser Gly SerGly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp AspPhe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Asp Ser Lys 85 90 95 Met PheGly Gln Gly Thr Lys Val Glu Val Lys 100 105 106 amino acids amino aciddouble linear protein 163 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile MetSer Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala SerSer Ser Ile Ser Tyr Met 20 25 30 His Trp Phe Gln Gln Lys Pro Gly Thr SerPro Lys Leu Trp Ile Tyr 35 40 45 Thr Thr Ser Asn Leu Ala Ser Gly Val ProAla Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr IleSer Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys His GlnArg Ser Thr Tyr Pro Leu Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu LeuLys 100 105 106 amino acids amino acid double linear protein 164 Asp IleGln Leu Thr Gln Ser Pro Ser Ser Met Ser Ala Ser Pro Gly 1 5 10 15 AspArg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Ile Ser Tyr Met 20 25 30 HisTrp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Trp Ile Tyr 35 40 45 ThrThr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 GlySer Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Met Gln Ala Glu 65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Thr Tyr Pro Leu Thr 85 90 95Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys 100 105 106 amino acids aminoacid double linear protein 165 Asp Ile Gln Met Thr Gln Ser Pro Ser ThrLeu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser AlaSer Ser Ser Ile Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly LysAla Pro Lys Leu Leu Ile Tyr 35 40 45 Thr Thr Ser Asn Leu Ala Ser Gly ValPro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu ThrIle Ser Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys HisGln Arg Ser Thr Tyr Pro Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val GluVal Lys 100 105 117 amino acids amino acid Not Relevant linear protein166 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 510 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Arg Ser 2025 30 Ala Ile Ile Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 3540 45 Gly Gly Ile Val Pro Met Phe Gly Pro Pro Asn Tyr Ala Gln Lys Phe 5055 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 6570 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Phe Cys85 90 95 Ala Gly Gly Tyr Gly Ile Tyr Ser Pro Glu Glu Tyr Asn Gly Gly Leu100 105 110 Val Thr Val Ser Ser 115 116 amino acids amino acid doublelinear protein 167 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala LysPro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr ThrPhe Thr Ser Tyr 20 25 30 Arg Met His Trp Val Lys Gln Arg Pro Gly Gln GlyLeu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Thr Gly Tyr Thr Glu TyrAsn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser SerSer Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Phe Glu Asp SerAla Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Gly Val Phe Asp Tyr Trp GlyGln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115 116 amino acidsamino acid double linear protein 168 Gln Val Gln Leu Gln Gln Ser Gly AlaGlu Val Ala Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys AlaSer Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Arg Met His Trp Val Lys Gln AlaPro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Thr GlyTyr Thr Glu Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr AlaAsp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu ArgSer Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Gly Val PheAsp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115 116amino acids amino acid double linear 169 Gln Val Gln Leu Val Gln Ser GlyAla Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys LysAla Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Arg Met His Trp Val Arg GlnAla Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser ThrGly Tyr Thr Glu Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Ile ThrAla Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser LeuArg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Gly ValPhe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 11580 base pairs nucleic acid single linear DNA 170 TGACTCGCCC GGCAAGTGATAGTGACTCTG TCTCCCAGAC ATGCAGACAT GGAAGATGAG 60 GACTGAGTCA TCTGGATGTC 8079 base pairs nucleic acid single linear DNA 171 TCACTTGCCG GGCGAGTCAGGACATTAATA GCTATTTAAG CTGGTTCCAG CAGAAACCAG 60 GGAAATCTCC TAAGACCCT 7979 base pairs nucleic acid single linear DNA 172 GATCCACTGC CACTGAACCTTGATGGGACC CCATCTACCA ATCTGTTTGC ACGATAGATC 60 AGGGTCTTAG GAGATTTCC 7985 base pairs nucleic acid single linear DNA 173 TGTCGACATC ATGGCTTGGGTGTGGACCTT GCTATTCCTG ATGGCAGCTG CCCAAAGTGC 60 CCAAGCACAG ATCCAGTTGGTGCAG 85

What is claimed is:
 1. A fusion protein comprising (a) a gelonin aminoacid sequence that has enzymatic activity and (b) a targeting sequencethat allows the internalization of said fusion protein, wherein saidtargeting sequence is an antibody, an antigen-binding portion of anantibody, a hormone, a lymphokine or a growth factor.
 2. The fusionprotein of claim 1, further comprising a linker sequence between saidgelonin sequence and said targeting sequence, wherein said geloninpossesses enzymatic activity, said antibody is capable of recognizingantigen and said hormone, lymphokine or growth factor is capable ofbinding to a cell that has a receptor for said hormone lymphokine orgrowth factor.
 3. The fusion protein of claim 2, wherein said linkersequence is that of SEQ ID NO. 56 or SEQ ID NO.
 57. 4. The fusionprotein of any one of claims 1, 2 and 3, wherein said targeting sequenceis an antibody.
 5. The fusion protein of any one of claims 1, 2 and 3,wherein said targeting sequence is an antigen-binding portion of anantibody.
 6. The fusion protein of claim 5, wherein said antigen-bindingportion of said antibody is an Fab.
 7. The fusion protein of claim 5,wherein, wherein said antigen-binding portion of said antibody is anFab′.
 8. The fusion protein of claim 5, wherein said antigen-bindingportion of said antibody is an F(ab′)₂.
 9. The fusion protein of claim5, wherein said antigen-binding portion of said antibody is an Fv. 10.The fusion protein of claim 5, wherein said antigen-binding portion ofsaid antibody has a single variable domain.
 11. The fusion protein ofclaim 5, wherein said antibody is a single-chain antibody.
 12. Thefusion protein of claim 5, wherein said fusion protein is multivalent.13. The fusion protein of any one of claims 1, 2 and 3 wherein saidtargeting sequence is a hormone.
 14. The fusion protein of any one ofclaims 1, 2 and 3, wherein said targeting sequence is an antibody. 15.The fusion protein of any one of claims 1, 2 and 3, wherein saidtargeting sequence is a growth factor.